6

Exploring Microbial Diversity and

PGPR Traits in Diversified Crop
Systems
SUMMARY

In sustainable agriculture, Plant Growth-Promoting Bacteria (PGPB) improve soil

health, stress resilience, and nutrient uptake. They produce beneficial compounds
like hormones and antibiotics, modify root exudates, and adjust to a variety of
environmental circumstances. Due to their ability to support crop vitality, these
microorganisms show promise for raising agricultural production in a variety of
farming conditions.Many isolates in different systems produced siderophores. 13 of
29 isolates in banana-based systems, 14 of 26 in citrus, 13 of 27 in guava, and 15 in
dragon fruit systems showed this capability. Different bacterial strains showed
differing capacities in the generation of Indole-3-acetic acid (IAA) and phosphate
solubilization in cropping systems that included bananas, citrus, guavas, and dragon
fruits. Under varying doses and incubation times of Triple Super Phosphate (TCP),
strains from each system showed unique capabilities. Utilizing molecular methods,
possible Plant Growth-Promoting Rhizobacteria (PGPR) were identified. The
genomic DNA was amplified by PCR and confirmed by gel electrophoresis to be
unique to the isolated bacterium. BLAST study found PGPR strains from banana,
citrus, guava, and dragon fruit-based systems using nucleotide sequences. The
analysis found Bacillus, Staphylococcus, and Fredinandcohnia. Phylogenetic
research utilizing ClustalW and MEGA X software determined these strains'
evolutionary relationships. Correlations were seen in dendrograms, suggesting
genetic diversity and relationships between certain PGPR strains and their relatives.
SEM demonstrated the morphologies of biofilms, highlighting colonization trends
and the advantages for agriculture. Genetic connections and capacities were
discovered through the analysis of genes and substances that create biofilms in a
variety of agricultural context.
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6.1. Introduction
The excessive use of synthetic fertilizers, which disturbs soil microbial ecosystems
vital to sustainable agriculture, makes it difficult to meet the world's food needs (Turk
et al. 2016; Zhang et al. 2020). Intensive farming practices and climate change
highlight the necessity of microbiological systems to improve agricultural
productivity (Kent et al. 2002; Rahman et al. 2018). In order to meet current demands
while preserving resources for future generations, sustainable agriculture strikes a
balance between environmental preservation and productivity (Shah et al. 2019;
Kremsa et al. 2021). Soil is essential for agricultural productivity because it promotes
nutrient cycling, organic matter decomposition, and the growth of microbial
populations that are critical to sustainability (Raj et al. 2019; Koshila Ravi et al. 2022;
Ouf et al. 2023). Microorganisms serve as natural soil engineers, improving soil
integrity and promoting healthier agricultural systems (Khatoon et al. 2020; Saccá et
al. 2017; Chethan Kumar et al. 2021). Plant-microbe interactions promote biodiversity
and crop resilience, which contributes to more sustainable agricultural methods and
productivity (Van Der Heijden et al. 2008; Prashar et al. 2014; Aislabie et al. 2013).
Soil biodiversity promotes nutrient cycling, disease resistance, and crop fitness, all of
which benefit ecological services and agricultural outputs (Shah et al. 2019; Schimel
et al. 2012; Jiang et al. 2022). Microorganisms in plants improve nutrient intake,
disease resistance, and environmental adaptation (Vishwakarma et al., 2020; Chauhan
et al. 2023). Plant growth is influenced by symbiotic connections, such as those
between arbuscular mycorrhizal fungi and nitrogen-fixing bacteria (Sarsaiya et al.
2020; Chauhan et al. 2021). Plant Growth-Promoting Bacteria (PGPB) provide
environmentally friendly treatments that improve soil health and agricultural output
(Kiruba N and Saeid, 2022; Morrissey et al., 2004; da Silva et al., 2021). They
promote complex microbial communities in the rhizosphere, which aids soil processes
and sustainable agriculture (Hawkes et al., 2007; Trivedi et al., 2020; and Arora et al.,
2013). Root exudates, which contain diverse nutrients and secondary metabolites,
nourish the rhizosphere, promoting microbial activity and soil stability (Afridi et al.,
2023; Odelade et al., 2019). Plant roots influence the rhizosphere environment by
attracting beneficial bacteria and increasing microbial populations (Di Benedetto et al.
2017).

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Plant Growth-increasing Bacteria (PGPB) regulate nitrogen supply and hormones,

increasing growth and nutrient absorption (Pattnaik et al. 2019; Sayyed et al. 2012).
PGPB produce antibiotics and siderophores, which reduce the impact of
phytopathogens (Saeed et al. 2021). The ACC deaminase enzyme in PGPB promotes
plant growth during stress by controlling ethylene levels (Muthukumar et al. 2016;
Bharti et al. 2019; Jha et al. 2021). Phosphorus, a critical plant nutrient, necessitates
management measures to improve accessibility due to its involvement in plant
metabolism (Karri et al. 2021). While synthetic phosphorus fertilizers increase
nutrient availability, they can cause eutrophication and disturb microbial diversity
(Qarni et al. 2021). To address these concerns, researchers are looking into microbes
that can enzymatically degrade calcium phosphates (Elhaissoufi et al. 2022).
Phosphate-solubilizing rhizobacteria in biofertilizers increase phosphate availability,
supporting sustainable agriculture (Mukherjee et al. 2019). Siderophores produced by
microorganisms increase iron availability in soils, improving plant health and disease
resistance (Singh et al. 2018; Zhang et al. 2019). They control plant pathogen
proliferation and stimulate plant development by increasing iron availability, which
leads to higher crop yields (Zhang et al. 2019; Saha et al. 2016). However, Potassium
(K) and zinc (Zn) are required for plant growth, but insufficient amounts might result
in lower yields and susceptibility to diseases (Liu et al. 2018). Soil bacteria perform
an important role in solubilizing these nutrients, increasing their availability to plants
(Sattar et al. 2019). Potassium-solubilizing bacteria found in a variety of settings,
including mica mines, have been shown to be capable of freeing potassium. Zinc
deficiency has an impact on essential plant processes, but microbial communities can
improve Zn supply in the rhizosphere, which benefits plant health (Hakim et al. 2021;
Hajiboland et al. 2012; Gondal et al. 2021). The 16S rRNA approach is critical for
identifying soil microbial diversity, providing insights into phylogeny and taxonomy
(Armougom et al. 2009; Rosselló-Mora et al. 2016). This method, together with
DNA-DNA similarity assessments, allows for more accurate classification of bacterial
strains (Janda et al. 2007). Using 16S rRNA sequences as housekeeping genes, PCR
amplification followed by amplicon sequencing allows for accurate genus-level
identification (Clarridge et al. 2004). However, while the genus identification
accuracy is over 90%, specificity drops to 65-83% at the species level (Karlsson et al.
2018). Despite the possibility of unclassified isolates, the 16S rRNA method is
nonetheless useful and efficient for identifying microbial strains
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6.2. Materials and Methods

6.2.1. Site description

Sample of soil taken from the semi-arid Mirzapur district in Eastern Uttar Pradesh.
The research region for Mirzapur is located roughly between latitudes 24°52.6213N
and 24°54.5975'N and longitudes 082°49.7966'E and 082°50.2868'E. Situated at an
elevation of 305.0 meters above sea level is the study area. In the Rajgarh region, red
lateritic soil and sandy loams make up the majority of the soil. A year-long thorough
investigation was conducted prior to choosing the cropping system. Adjacent plots in
the Rajgarh region were selected in order to compare different cropping strategies
with monocropping. Two conditions had to be met before choosing a cropping field.
First of all, farmers in the area had to have a minimum landholding of 0.5 hectares
and implement crop diversification schemes. Second, it made proactive management
of agricultural diversification initiatives necessary.

6.2.2. Soil sample collection

The goal of this study was to extract and purify bacteria from various cropping
systems, such as banana (BBC), citrus (CBC), guava (GBC), and dragon fruit (DBC),
up to 15 cm depth using hand trowels. The samples were gathered in bags and brought
to the lab stored at 4°C in arefrigerator for further studies. The major purpose was to
examine the existence and variety of bacterial communities in monocropping and
varied cropping soils, which would provide insights into plant-microbe connections
and the impact of cultivation strategies on soil microbiota. Comparing these various
systems could reveal patterns of microbial diversity and interactions that are
important for sustainable agricultural methods. The analytical approach enabled a
comparison of the existence and diversity of bacterial communities in monocropping
and varied cropping soils, potentially revealing insights into plant-microbe
connections and the impact of cultivation strategies on soil microbiota.

6.2.3. Isolation of bacteria from soil sample

The extraction of bacterial strains from soil samples was conducted using a serial
dilution approach. A soil sample weighing 10g was combined with a sterile saline
solution and subjected to agitation using an orbital shaker. A soil suspension with a
volume of 0.1 mL was evenly distributed over Nutrient Agar Medium plates in order
to isolate non-specific bacteria. To obtain pure single colonies, distinct and rapidly

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proliferating colonies were carefully chosen and subsequently streaked onto fresh
plates. The strains were evaluated for their Plant growth-promoting (PGP)
characteristics and subsequently preserved at a temperature of -20°C using glycerol
stocks for potential future utilization.

6.2.4. Biochemical and Morphological analysis of bacterial isolate

In accordance with recommendations from Bergey's Manual of Systematic
Bacteriology (Kreig and Holt 1984), the biochemical and morphological
investigations of particular bacterial isolates involved in vitro experiments to confirm
their biochemical features. The purpose of these tests was to validate the features of
the isolates.

6.2.5. Bacterial preliminary identification

Based on the isolated strains' morphological, cultural, and staining traits, the study
distinguished between them. The bacterial isolates were cultured on Nutrient Agar
Medium (NAM) plates for a duration of 48 hours. During this period, many properties
of the colonies were examined, including color, elevation, surface texture, margin
appearance, optical characteristics, and growth rate. The examination of cellular
morphology was conducted utilizing light microscopy, wherein the cell's shape and
Gram reaction were evaluated. Subsequently, the isolates were subjected to Gram
reaction analysis by light microscopy. The objective of the study was to classify the
isolates according to their distinct characteristics, including color, texture, and pace of
growth. The results offer significant insights on the differentiation and diversification
of bacterial isolates.

6.2.6. Gram staining

The Gram staining technique was first described by Gram in 1884 and was used to
identify the Gram type of bacterial isolates. A glass slide was utilized to distribute a
colony of bacterial isolates that were 24 hours old. The slide was then air-dried and
subsequently heat-fixed. The crystal violet stain was administered, allowed to
incubate for a duration of one minute, and then excess stain was eliminated using
tissue paper. Subsequently, the slide was subjected to a water rinse, followed by the
addition of Gram iodine, and left undisturbed for a duration of 30 seconds.
Subsequently, the stain was subjected to ethanol washing and subsequently subjected
to counter-staining with safranin for a duration of one minute. The slide was

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subsequently analyzed under oil immersion utilizing a Nikon E200 microscope. The
utilization of this extensive staining procedure facilitated the evaluation of the Gram
features of the bacterial isolates. The procedure encompassed a series of sequential
steps, which entailed the elimination of surplus stain, rinsing with water, and
subsequent counter-staining utilizing safranin.

6.2.7. In vitro testing of bacterial isolates for PGP characteristics

Bacterial isolates were initially tested for Plant Growth Promoting (PGP)
characteristics such as phosphate, potassium, and zinc solubility and the production of
chemicals such as Indole-3-Acetic Acid (IAA), siderophores, and ammonia.
Following confirmation of these characteristics, the isolates were subjected to a more
comprehensive examination. Secondary qualitative screening investigated the isolates'
ability to solubilize these nutrients, while secondary quantitative screening quantified
the extent of phosphate and potassium solubilization as well as IAA and siderophores
production levels. This quantitative analysis offered accurate data on the amount of
these compounds produced by the bacteria, allowing researchers to better appreciate
their potential effectiveness in boosting plant growth. The primary screening
functioned as a selection process based on basic PGP properties, whereas the
secondary screening attempted to provide a full understanding of the effectiveness
and quantity of these qualities exhibited by the selected bacterial isolates. This
comprehensive assessment is critical in identifying microorganisms with high PGP
potential for agricultural applications.

6.2.8. Qualitative analysis of Phosphate solubilization

The Phosphate Solubilization Efficiency (PSE) of isolated bacterial strains was
assessed in the study by inoculating 5 µL of a rhizobacterial broth culture onto
Pikovskaya's Agar plates. These plates were supplemented with tricalcium phosphate
(TCP) as an insoluble inorganic phosphate source. The bacterial samples were
subjected to incubation at a temperature of 28±2 °C. Subsequently, the samples were
inspected on the 3th, 5th, and 7th Day After Incubation (DAI) in order to identify the
presence of clearing zones surrounding the bacterial colonies. The calculation of the
Phosphate Solubilization Index (PSI) involved the measurement of the combined
diameter of the colony and the individual bacterial colony. This measurement was
performed according to the formula established by Edi-Premono et al. (1996). The

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utilization of this approach facilitated a quantitative evaluation of the efficacy of the

bacterial strains in the process of phosphate solubilization.

Total diameter (colony + halo zone)

Phosphate Solubilization Index (PSI) =
Diameter of colony

6.2.9. Quantitative analysis of Phosphate solubilization

The study quantitatively examined the solubilization of phosphate in a liquid media
using Barton's (1948) approach. The bacterial cell suspensions were calibrated to a
concentration of 108 colony-forming units per milliliter (CFU mL-1) using a
spectrophotometer. A 50 mL volume of Pikovskaya medium was inoculated with a
very effective isolate and subjected to incubation at a controlled temperature of 28±2
°C for a duration ranging from 24 to 96 hours. The quantification of solubilized
inorganic phosphate was conducted within a time frame of 24 to 72 hours. The culture
broth samples underwent centrifugation, and then, their supernatants were combined
with Barton reagent. The measurement of absorbance at a wavelength of 430 nm was
conducted using a UV-VIS Spectrophotometer following a duration of 10 minutes.
The aforementioned procedure was conducted three times in order to measure the
amount of solubilized phosphorous, hence offering valuable information regarding the
kinetics of solubilization shown by the bacterial isolates. The findings provide
insights into the solubilization kinetics demonstrated by the bacterial isolates.

6.2.9.1. Preparation of the stock solution and standard curve of phosphate

To prepare a working solution of 30 µg phosphorus mL-1, 0.2195 g of dry KH2PO4
were dissolved in distilled water to create a phosphorus stock solution. A standard
curve was generated by quantifying quantities of the working solution into 50 mL
Volumetric Flasks, each containing 2.5 mL of Barton reagent. A solution devoid of the
working solution was produced. The measurement of absorbance was conducted at a
wavelength of 430 nm using a UV-VIS Spectrophotometer following a duration of 10
minutes. A standard curve was generated by graphing the amounts of phosphorus
against their respective absorbance values at a wavelength of 430 nm. The curve
presented herein serves as a valuable tool for estimating the quantities of phosphorus
in subsequent samples, utilizing their respective absorbance values as a reference. The
procedure facilitates the measurement of phosphorus concentration in successive
samples.

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6.2.9.2. Qualitative analysis of Potassium solubilization

The study used Aleksandrov agar medium with insoluble potassium alumino silicate
to test the ability of bacterial isolates to dissolve potassium. The bacterial culture
droplets were deposited onto the medium and subjected to incubation at a temperature
of 28±2 °C for a duration of three days. A distinct area devoid of bacterial growth was
detected surrounding the bacterial culture on the third, fifth, and seventh days
subsequent to injection. The calculation of the Potassium Solubilization Index (KSI)
involved the measurement of the combined horizontal and vertical diameters of both
the colony and the halo zone. This measurement allowed for a quantitative assessment
of the potassium solubilization effectiveness of the isolates. The observation of a
distinct area devoid of bacterial growth surrounding the culture site indicated the
possibility of potassium solubilization.

Total diameter (colony + halo zone)

Potassium Solubilization Index (KSI) =
Diameter of colony

6.2.9.3. Quantitative analysis of Potassium solubilization

The solubilization of potassium alumino silicate from a modified Aleksandrov broth
was evaluated using a Flame Photometer in this work. Bacterial suspensions were
introduced into the liquid medium, subjected to incubation, and thereafter transferred
to Eppendorf tubes to undergo centrifugation at a speed of 10,000 revolutions per
minute for a duration of 15 minutes. The quantification of potassium levels was
performed on a 1 mL sample of the supernatant, which was subsequently diluted with
distilled water to achieve a final volume of 10 mL. The utilization of this
comprehensive methodology facilitated the accurate measurement of potassium
solubilization resulting from potassium alumino silicate. The specimens were
collected on the 3rd, 5th, and 7th days after inoculation and subsequently underwent
centrifugation at a speed of 10,000 revolutions per minute for a duration of 15
minutes. The potassium concentrations were subsequently evaluated in the resultant
supernatant, which was diluted with distilled water. The chosen methodology
facilitated a quantitative assessment of solubilization by measuring the potassium
concentrations in the culture medium at designated time intervals.

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6.2.9.4. Qualitative analysis of Zinc solubilization

Using a modified Pikovskaya agar medium, which contains insoluble zinc oxide
(ZnO), the study aims to evaluate the solubilization ability of bacterial isolates. The
bacterial culture was applied as droplets onto the medium and thereafter subjected to
incubation at a temperature of 28±2 °C for a duration of 3-4 days. The study involved
conducting observations on the 3th, 5th, and 7th days after inoculation (DAI). The
presence of a distinct halo zone surrounding the bacterial culture spot served as an
indication of the isolate's ability to solubilize zinc. The determination of the Zinc
Solubilization Index (ZSI) involved the measurement of both the horizontal and
vertical diameters of the entire zone, which encompasses both the bacterial colony
and the halo zone. The efficacy of the isolate in facilitating the solubilization of zinc
was assessed using a calculation employing a designated formula. This work offers
significant insights into the capacity of bacterial isolates to enhance the solubilization
of zinc.

Total diameter (colony + halo zone)

Zinc Solubilization Index (ZSI) =
Diameter of colony

6.2.9.5. IAA (Indole-3-Acetic Acid) production

The utilization of the Salkowaski reagent was employed to investigate and quantify
the synthesis of Indole-3-acetic acid (IAA) by bacterial isolates, as evidenced by a
visible alteration in color from yellow to pink. A modified version of Tang and
Bonner's (1948) method, which called for 48 hours of bacterial suspension in nutrient
broth incubation, was used to assess the number of positive isolates. The
concentration of IAA in 1 mL of the resultant supernatant was determined by the
addition of ortho-phosphoric acid and Salkowaski reagent. A blank was made in the
same manner using nutrition broth. Both samples were subjected to incubation in a
lightless environment for a duration of 30 minutes, following which their absorbance
at a wavelength of 535 nm was quantified utilizing a UV-VIS Spectrophotometer. The
quantification of indole-3-acetic acid (IAA) production was performed using the
linear regression equation obtained from an IAA standard curve. The rhizobacterial
isolates that produced the highest amount of indole-3-acetic acid (IAA) were
subjected to additional analysis in order to investigate the impact of incubation
duration on their IAA production. The objective of this study was to employ a precise
measurement technique for assessing indole-3-acetic acid (IAA) synthesis, as well as

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investigate the impact of varying incubation durations on the levels of IAA in

bacterial isolates.

6.2.9.6. Preparation of stock solution and standard curve of IAA

The stock solution of Indole-3-acetic acid (IAA) used in this study had a
concentration of 0.1 mg mL-1, prepared in a solvent consisting of 50% ethanol. A
solution containing 10 mg of indole-3-acetic acid (IAA) was prepared by dissolving it
in a small quantity of 50% ethanol, followed by dilution with distilled water to a final
volume of 100 mL. The standard curve was generated by filling test tubes with
varying volumes of the IAA standard stock solution, specifically 0.05, 0.1, 0.15, 0.2,
0.3, 0.4, 0.5, 0.8, and 1 mL. Following the addition of 2 mL of Salkowski reagent and
1-2 drops of orthophosphoric acid, the IAA solutions were then supplemented to a
final volume of 3 mL using nutrient broth. A blank solution was prepared by
combining 1-2 drops of orthophosphoric acid and 2 mL of Salkowski reagent with 1
mL of nourishing broth in the absence of the indole-3-acetic acid (IAA) stock
solution. Both mixtures were subjected to incubation at ambient temperature for a
duration of 30 minutes under conditions of darkness. The absorbance of the sample
was measured at a wavelength of 535 nm. The construction of the standard curve
involved plotting the concentration of IAA solution on the X-axis and the
corresponding absorbance at 535 nm on the Y-axis (see Appendix VB). The utilization
of a standardized curve facilitated the quantification of following samples by means
of comparing their absorbance values at 535 nm to established concentrations of IAA.

6.2.9.7. Qualitative estimation of siderophore production

Using Chrome Azurol S (CAS) agar medium plates and the methodology described by
Schwyn and Neilands (1987), the study investigated the potential of bacterial isolates
for siderophore synthesis. The bacterial isolates were subjected to cultivation in a
nutrient broth medium for a duration of 24 hours, employing an orbital shaker set at a
temperature of 28±2 °C. Subsequently, a volume of 5 μL of a bacterial suspension was
applied onto the plates, followed by incubation at a temperature of 28±2 °C for a
duration of 7 days. The examination conducted toward the conclusion of the
incubation period unveiled the existence of a yellow-orange halo region encircling the
bacterial spot, thereby signifying the synthesis of siderophores. The halo zone
exhibited by the bacterial isolates acted as a visual indicator for the generation of

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siderophores. This study offers significant insights into the potential of bacterial
isolates in the production of siderophores.

6.2.9.8. Ammonia Production

The procedure for assessing ammonia production entails introducing recently
cultivated bacterial isolates into a solution of peptone broth, followed by incubation at
a temperature of 28±2 °C for a duration of 72 hours. Following the incubation period,
a volume of 0.5 mL of Nessler reagent is introduced into the broth, and subsequent
alterations in color are carefully monitored and documented. The methodology
employed in this study adheres to the assessment protocol established by Cappuccino
and Sherman (1992) for quantifying ammonia output. Subsequently, the obtained
outcomes are employed to ascertain the synthesis of ammonia.

6.2.9.9. HCN Production

The study aims to assess the generation of hydrogen cyanide (HCN) by bacterial
isolates using a nutritional medium containing 4.4 g L-1 glycine. Subsequently, the
medium was injected with bacteria and securely attached on Petri plates.
Subsequently, filter paper disks were positioned onto the lids of the plates, thereby
being subjected to the bacterial streaks. The plates were securely sealed using
parafilm and subjected to incubation at a temperature of 28±2 °C for a period of 5 to 7
days. The process of glycine degradation results in the synthesis of hydrogen cyanide
(HCN), a chemical known for its high volatility. The reaction between hydrogen
cyanide (HCN) and picric acid in the presence of sodium carbonate (Na2CO3) results
in a progressive color transformation on the filter paper. Initially, the filter paper
exhibits a deep yellow color, which then transitions to orange, followed by an orange-
brown shade, and ultimately reaches a dark brown coloration. In the event that the test
produces negative outcomes, it may be observed that the filter paper maintains its
intense yellow color despite the presence of bacterial proliferation.

6.2.9.9.1. Salinity Sensitive Test

Individually, fresh 48-hour-old 0.5 mL PGPR isolates were inoculated into 100 mL
culture tube containing 20 mL LB broth (Annexure I) with various NaCl
concentrations (1, 3, and 5%). After 48 hours of incubation at 37oC, the abundance of
growth was evaluated at 620 nm. The results were rated as positive or negative in
terms of growth. Bacterial isolates can be cultured or propagated on media in which

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the concentration of NaCl is purposely altered to specific percentages. The isolates'

growth patterns, viability, and metabolic activities can then be investigated and
compared at various salt concentrations to determine their tolerance or sensitivity to
differing degrees of salinity.

6.2.9.9.2. The pH Sensitive Test

The growth or survival of bacterial isolates in response to different pH levels can be
investigated using the pH-sensitive test. This experimental procedure entails
subjecting several cultures to varying pH levels, ranging from acidic to alkaline
settings. Observations are conducted in order to ascertain the isolates' capacity to
tolerate specific pH values, thereby discerning the optimal, suboptimal, and maximal
pH ranges conducive to their development or survival. The objective of the
experiment is to evaluate the bacterial organisms' ability to adapt to different pH
levels, specifically pH values of 5, 8, and 9. The adjustment of the solution is
accomplished through the utilization of Succinic acid buffer solutions, while the
bacterial cultures are introduced into modified broths. The assessment of growth
patterns across varied pH settings using the absorbance at a 600 nm wavelength offers
valuable insights into the adaptive mechanisms of bacterial species in response to
fluctuations in pH conditions.

6.2.9.9.3. Temperature Sensitive Test

The temperature-sensitive assay is a technique employed to evaluate the proliferation
or viability of bacterial isolates across varying temperature gradients. This
experimental procedure entails subjecting the cultures to a range of temperatures,
spanning from a lower value of 4°C to higher values of either 25°C, 42°C and 55°C.
Observations are conducted during a specified timeframe in order to ascertain the
most favorable, less favorable, and highest temperatures for the purposes of
development or survival. The present experiment facilitates the comprehension of the
thermal thresholds and inclinations of bacterial isolates, hence offering significant
insights into their ecological adaptations. This analysis aids in the determination of
the most favorable, less favorable, and highest temperature ranges that facilitate the
development or survival of the isolates, hence offering valuable insights on their
thermal preferences and constraints.

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6.2.9.9.4. Potential PGPR bacterial strain genomic DNA extraction

The method used in this work, which was inspired by Araujo et al. (2002), is to
extract genomic DNA from bacterial isolates. The procedure is subjecting actively
proliferating bacteria in Luria-Bertani (LB) broth to centrifugation at a speed of 6,000
rpm per minute for a duration of 15 minutes while maintaining a temperature of 4°C.
The pellet obtained is subjected to a wash step using 1X TE buffer, followed by
resuspension in a solution containing 10 mL of 1X TE buffer and 250 μL of SDS.
Next, a volume of 20 μL of Proteinase K is introduced into the mixture, which is
subsequently subjected to incubation at a temperature of 37°C for a duration of 1
hour. A total volume of 900 μL of a 5M NaCl solution and 750 μL of a 5% CTAB
solution were combined and subjected to an incubation period of 20 minutes at a
temperature of 65°C. Following the process of cooling to ambient temperature, a
volume of 10 mL of Chloroform: Isoamylalcohol (24:1) is introduced, and
subsequently subjected to manual mixing for a duration of 10 minutes using rotary
motion. The resulting mixture is then subjected to centrifugation at a speed of 12,000
revolutions per minute for a period of 20 minutes while maintaining a temperature of
4°C. To extract DNA, move the upper aqueous layer to a new tube, mix
phenol:chloroform:isoamyl alcohol (25:24:1), add 10 μL of RNase A, and centrifuge
for 10 minutes at 10,000 rpm. Three to four repetitions of this method are necessary to
remove the white coating. After that, the top aqueous layer is moved to a
microcentrifuge tube that has been sanitized. Cold isopropanol is then added, and the
mixture is stirred until DNA precipitates. Centrifugation is used to extract the DNA; it
takes 12 minutes at 4 ºC and 10,000 rpm. After air drying and two 70% ethanol rinses,
the pellet is suspended in 30–50 µL of 1X TE (Tris-EDTA) buffer. Overnight, the
DNA is kept at -20 ºC. This process makes it easier to gather and store the extracted
DNA at the end.

6.2.9.9.5. Agarose gel electrophoresis

Genomic DNA was subjected to gel electrophoresis analysis. Boiling a 0.8% Agarose
solution in 1X TAE buffer was followed by the addition of 5 μL of Ethidium bromide,
pouring the mixture into a tray, taping it shut, and inserting a comb to make wells.
After allowing the gel to set, the sealing tape and comb were taken off. For further
electrophoresis examination, the casting tray containing the gel was put in a buffer
tank that was filled with recently made 1X TAE buffer. After that, the gel was put

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inside the tank, and the sealing tape and comb were taken out. For additional
investigation, the gel was subsequently put within a buffer tank filled with recently
made 1X TAE buffer.

6.2.9.9.6. Qualitative and quantitative analysis of DNA

The gel electrophoresis procedure consisted of introducing a DNA ladder into the
initial well, and subsequently introducing bacterial isolate DNA samples into the
second to eight wells. The loading dye was added to each sample in a volume of 2 μL,
and thereafter loaded into their corresponding wells. In order to do a quantitative
estimation, a well was loaded with a known concentration of λ uncut DNA/Hind III.
The gel was subjected to an electrical potential of 80 volts for a duration of 30
minutes, resulting in the migration of the DNA to a position halfway through the gel
tray. Following the completion of the run, the gels were subjected to examination and
analysis utilizing a UV transilluminator.

6.2.9.9.7. 16S rRNA amplification

The process of DNA amplification was conducted by utilizing universal Forward and
Reverse primers that specifically targeted the 16S rRNA gene. This amplification was
carried out in a BioRad T100 thermocycler. The primers were produced as a stock
solution by dissolving them in sterile TE buffer with a pH of 7. To achieve a
concentration of 100 µM, a dilution process was employed by adding 10 μL of the
stock primer solution to TE buffer. The amplification procedure entailed the
utilization of a reaction mixture with a volume of 50 μL. This mixture comprised 25
μL of a 2X PCR Master Mix solution, 2 μL of both the Forward and Reverse primers,
2 μL of the DNA template, and 19 μL of Milli Q water. The amplification procedure
consisted of a reaction mixture with a volume of 50 μL. The thermocycler procedure
commenced with an initial denaturation phase at a temperature of 95°C for a duration
of 3 minutes, succeeded by 30 amplification cycles at the same temperature, with each
cycle lasting 1 minute. The annealing step was conducted at a temperature of 58°C for
a duration of 30 seconds, followed by an extension step at 72°C for a period of 1
minute. The concluding phase involved a polymerization process conducted at a
temperature of 72°C for a duration of 8 minutes.

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6.2.9.9.8. Touchdown PCR

The determination of the annealing temperature, which is essential for the binding of
primers to DNA, was conducted by the utilization of a touchdown PCR technique.
The experimental procedure consisted of an initial denaturation step at a temperature
of 95°C for a duration of 3 minutes, followed by a series of 30 amplification cycles at
a temperature of 95°C for a duration of 1 minute each.

Table 6.1. The reaction mixture employed for the amplification of the 16s rRNA gene.

Serial No. Components (1X) Volume (μL)

1 2X Mater Mix solution 25
2 Reverse Primer 2
3 Forward Primer 2
4 Template DNA 2
5 Millipore Water 19
6 Total reaction Mixture 50

The annealing temperature was varied between the range of 50°C to 60°C for a
duration of 30 seconds, after which an extension phase was conducted at a constant
temperature of 72°C for a period of 2 minutes. The temperature of the final
polymerization process was held at 72°C for a duration of 8 minutes. The detailed
description of reaction mixture mentation in Table 6.1.

6.2.9.9.9Extraction of amplified DNA from gel

The amplified DNA fragments were removed from a gel using a Gel Doc platform
using a sterilized blade. The gel that was obtained was subsequently resuspended in 1
mL of SET buffer using a 1.5 mL Eppendorf tube and then subjected to a 50°C water
bath. The mixture was subjected to vortexing at regular intervals of 2-3 minutes until
complete dissolution of the gel slice occurred. The dissolved sample was introduced
into a column contained within the Pure Extract Spin PCR Cleanup/Gel Extraction kit
and subjected to centrifugation at a speed of 12,000 revolutions per minute for a
duration of 1 minute. The remaining substance within the Eppendorf tube was
disposed of. Subsequently, the column was transferred to a new collection tube,
followed by the addition of 700 μL of SET-3 buffer. The resulting mixture was then
subjected to centrifugation at a speed of 10,000 revolutions per minute for a duration
of 1 minute. The waste material derived from the collection tube was disposed of. To
make sure all of the SET-3 buffer was removed, the empty column was centrifuged

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for two minutes at 11,000 rpm. Subsequently, the column was transferred into a new
Eppendorf tube and supplemented with 15-50 µL of SEB buffer. The mixture was
allowed to incubate for a duration of 1 minute, followed by centrifugation at a speed
of 11,000 revolutions per minute for a period of 10 minutes. The procedure facilitated
the thorough elution of the amplified DNA from the column, effectively gathering it
within the Eppendorf tube for subsequent utilization.

6.2.9.9.9.1. Analysis of 16S rRNA gene sequence of PGPR isolates

The ABI Prism Terminator Cycle Sequencing Ready Reaction Kit was used to carry
out sequencing reactions, which were then exposed to electrophoresis to evaluate the
resulting products. The electrophoresis method was carried out in Anuvanshiki (OPC)
Pvt. Ltd. utilizing an Applied Biosystems (Model 3100) automated sequencer. This
approach allowed for the inspection and identification of DNA sequence data from
samples, therefore speeding up the DNA analysis process. The process of sequence
editing and assembly was carried out using BioEdit Version 7.0.5.3. The sequences
were subsequently converted into FASTA format and underwent BLAST analysis
utilizing the GenBank database sourced from the National Center for Biotechnology
Information (NCBI). The BLAST tool was employed to identify commonalities across
nucleotide sequences, thereby establishing their identity by comparison with the most
closely related entries in the database. The accession numbers for all sequences were
acquired by the submission of said sequences to the GenBank database, operated by
the National Center for Biotechnology Information (NCBI). The procedure entailed
the manipulation and arrangement of sequences with BioEdit Version 7.0.5.3.

6.2.9.9.9.2. Data analysis and construction of Phylogenetic tree

The analysis of the 16S rRNA gene sequences of selected PGPR isolates was
conducted in this study using the NCBI BLAST program, employing both non-
redundant (nr) and microbiological databases. The phylogenetic analysis was
conducted with the Clustal W algorithm integrated into the software, and the
alignment of sequences was accomplished using CLUSTAL X software. The
assessment of the reliability of internal branches was conducted using a bootstrap
phylogenetic test consisting of 1,000 replicates. The construction of the phylogenetic
tree involved the utilization of 16S rDNA sequences obtained from several eubacteria,
which were acquired from the National Center for Biotechnology Information
(NCBI). The evolutionary distances were computed with the neighbor-joining

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approach as proposed by Saitou and Nei (1987) and implemented through MEGA 4.0
software. The tree file was subjected to analysis using the treeview software.

6.3. Results and discussion

6.3.1. Morphological and biochemical characterization of PGPR isolates
Rhizobacterial isolates obtained from a range of diversified cropping systems were
examined in this investigation. These systems included banana (BBC), citrus (CBC),
guava (GBC), and Dragon-fruits (DBC) based systems. The CBC isolates were
classified into 26 groups (Table 6.3.), whereas the BBC isolates were classified into
29 groups (Table 6.2.). In contrast, the DBC isolates were classified into 25 groups
(Table 6.5.), whereas the GBC isolates were classified into 27 groups (Table 6.4.). The
isolates were categorized as follows: banana-derived (DBC), Guava-derive (GBC),
citrus-derived (CBC), and dragon fruit-derived (BBC). The study's findings offer
significant contributions to the understanding of rhizobacterial diversity and the
potential health advantages associated with various cultivation systems.

6.3.2. Morphological characterization of isolated rhizobacterial strain

The study characterizes rhizobacterial isolates across various cropping systems,
revealing their potential for enhancing plant health and disease resistance. Tables 6.2.
to 6.5. outline isolates' classifications by Gram reaction in banana, citrus, guava, and
dragon fruit systems. Figures 6.1 and 6.2. visually depict the diverse cellular shapes
and colony morphologies observed. This research provides valuable insights into
agricultural practices, aiding in the development of biocontrol methods and
environmental remediation strategies.

6.3.3. pH Sensitive test of isolated rhizobacterial strain of diversified cropping

system
Understanding pH sensitivity in rhizobacterial strains is critical for nutrient
absorption, plant health, and soil productivity, encouraging environmentally friendly
agriculture methods (Fasusi et al., 2023). Table 6.6 depicts the varied growth trends of
rhizobacterial isolates from banana-based systems across pH levels. Similarly, Tables
6.7 to 6.9 describe growth characteristics in citrus, guava and dragon fruits cropping
systems, respectively. Figure 6.1 demonstrates the pH sensitivity of rhizobacterial
isolates in banana cultivation, which helps with agricultural diversification and
sustainable methods.

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Table 6.2. Cultural and morphological characteristic of isolates microorganism from Banana Based cropping system.

Serial Culture Shape Size Margin Elevation Consistency Color Surface Opacity Odour Gram’s Arrangement
no name reaction
1 BBC1 Irregular Medium Entire Raised Moist Creamy Smooth Opaque No GPR Singular, Chain
2 BBC 2 Round Big Entire Raised Moist White Slimy Opaque No GPR Singular, Chain
3 BBC 3 Round Medium Entire Flat Moist White Smooth Opaque No GPR Singular, Chain
4 BBC 4 Round Medium Entire Raised Moist Off-white glistening Opaque No GPR Singular, Chain
5 BBC 5 Round Big Entire Raised Creamy White Smooth Opaque No GPR Singular, Chain
6 BBC 6 Round Medium Regular Undulate Moist White Smooth Translucent No GPR Singular, Chain
7 BBC 7 Round Medium Entire Raised Moist Creamy Slimy Opaque No GPR Singular, Chain
8 BBC 8 Round Big Entire Convex Moist White Smooth Opaque No GPR Singular, Chain
9 BBC 9 Round Medium Entire Raised Moist Yellow Smooth Opaque No GNR Singular, Chain
10 BBC 10 Round Medium Regular Raised Moist White glistening Opaque No GPR Singular, Chain
11 BBC 11 Round Big Entire Raised Sticky White Smooth Opaque No GPR Singular, Chain
12 BBC 12 Round Medium Entire Convex Moist Creamy Slimy Opaque YES GPR Singular, Chain
13 BBC 13 Round Medium Entire Raised Moist White Smooth Opaque No GPR Singular, Chain
14 BBC 14 Round Big Entire Raised Moist White Smooth Translucent No GPR Singular, Chain
15 BBC 15 Round Medium Entire Flat Moist Brown Slimy Opaque No GNR Singular, Chain
16 BBC 16 Round Medium Entire Raised Moist White Smooth Opaque No GPR Singular, Chain
17 BBC 17 Round Big Regular Convex Dry White Smooth Opaque No GPR Singular, Chain
18 BBC 18 Round Medium Entire Raised Moist Off-white Slimy Opaque No GPR Singular, Chain
19 BBC 19 Round Medium Entire Raised Moist Yellow Smooth Translucent No GPR Singular, Chain
20 BBC 20 Round Big Entire Raised Moist White Smooth Opaque No GNR Singular, Chain
21 BBC 21 Round Big Entire Convex Dry White Slimy Opaque No GPR Singular, Chain
22 BBC 22 Round Medium Entire Raised Moist Yellow Smooth Opaque No GNR Singular, Chain
23 BBC 23 Round Medium Entire Raised Moist White glistening Opaque No GPR Singular, Chain
24 BBC 24 Round Big Regular Raised Sticky White Smooth Translucent No GPR Singular, Chain
25 BBC 25 Round Medium Entire Flat Dry White Smooth Opaque No GNR Singular, Chain
26 BBC 26 Round Medium Entire Raised Moist Off-white glistening Opaque YES GPR Singular, Chain
27 BBC 27 Round Big Regular Raised Creamy White Smooth Opaque No GNR Singular, Chain
28 BBC 28 Round Medium Entire Raised Moist White Slimy Translucent No GPR Singular, Chain
29 BBC 29 Round Big Entire Raised Moist White Smooth Opaque No GPR Singular, Chain

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Table 6.3. Colonial and morphological characteristics of selected microorganism from the citrus-based cropping system.

Sl.No. Culture shape size Margin Elevation Consistency Color Surface Opacity Odour Gram’s Arrangement
name reaction
1 CBC1 Round Small Regular Raised Moist White Smooth Opaque No GNR Singular
2 CBC2 Round Small Regular Raised Moist White Smooth Opaque No GNR Singular
3 CBC3 Round Large Regular Flat Creamy Off-white Slimy Opaque No GNR Singular, Chain
4 CBC4 Round Medium Entire Raised Moist White Smooth Translucent No GPR Singular, Chain
5 CBC5 Round Small Regular Raised Moist Creamy Smooth Translucent YES GNR Singular
6 CBC6 Round Small Entire Convex Dry White glistening Translucent No GPR Singular
7 CBC7 Round Small Regular Raised Moist White Smooth Opaque No GNR Singular
8 CBC8 Round Medium Regular Undulate Moist Yellow Smooth Opaque No GPR Singular, Chain
9 CBC9 Round Medium Regular Raised Moist White Slimy Translucent No GNR Singular
10 CBC10 Round Large Regular Flat Sticky Creamy Smooth Translucent No GPR Singular
11 CBC11 Round Small Entire Raised Dry White Smooth Opaque No GNR Singular
12 CBC12 Round Small Regular Raised Moist White Smooth Opaque No GPR Singular, Chain
13 CBC13 Round Small Regular Convex Moist yellow glistening Opaque No GNR Singular
14 CBC14 Round Small Regular Raised Dry White Slimy Translucent No GNR Singular, Chain
15 CBC15 Round Small Regular Raised Moist White Smooth Opaque No GNR Singular, Chain
16 CBC16 Round Small Regular Raised Moist White Smooth Opaque YES GNR Singular, Chain
17 CBC17 Round Large Regular Flat creamy Off-white Slimy Opaque No GNR Singular, Chain
18 CBC18 Round Medium Entire Raised Moist White Smooth Translucent No GPR Singular
19 CBC19 Round Small Regular Raised Moist Creamy Smooth Translucent No GNR Singular
20 CBC20 Round Small Entire Raised Moist White Smooth Opaque No GNR Singular
21 CBC21 Round Small Regular Raised Dry White Smooth Opaque No GPR Singular
22 CBC22 Round Small Regular Convex Moist yellow glistening Opaque No GNR Singular, Chain
23 CBC23 Round Small Regular Raised Dry White Slimy Translucent YES GPR Singular
24 CBC24 Round Small Regular Raised Moist Creamy Smooth Translucent No GNR Singular, Chain
25 CBC25 Round Small Entire Convex Moist White glistening Translucent No GPR Singular
26 CBC26 Round Small Regular Raised Dry White Smooth Opaque No GNR Singular

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Table 6.4. Colonial and morphological characteristics of selected microorganism from the Guava -based cropping system

Sl. Culture shape size Margin Elevation Consistency Color Surface Opacity Odour Gram’s Arrangement
No. name reaction
1 GBC1 Round Pinpoint Rhizoid Raised Moist White Smooth Translucent No GPR Singular, Chain
2 GBC2 Round Small Irregular Flat Creamy White Smooth Opaque No GPR Singular
3 GBC3 Round Small Regular Raised Moist Creamy Smooth Opaque No GPR Singular
4 GBC4 Round Small Regular Raised Moist Creamy Slimy Opaque No GNR Singular
5 GBC5 Round Large Irregular Flat Moist White glistening Translucent No GPR Singular, Chain
6 GBC6 Round Small Regular Raised Sticky White Smooth Opaque No GNR Singular, Chain
7 GBC7 Round Small Rhizoid Convex Moist Yellow Smooth Opaque No GPR Singular, Chain
8 GBC8 Round Medium Regular Raised Moist White Smooth Opaque No GPR Singular, Chain
9 GBC9 Round Small Regular Raised Moist Brown Slimy Translucent No GPR Singular
10 GBC10 Round Large Rhizoid Convex Dry White Smooth Translucent No GNR Singular
11 GBC11 Round Small Regular Raised Moist White Smooth Opaque No GNR Singular
12 GBC12 Round Pinpoint Irregular Raised Moist Creamy glistening Opaque No GNR Singular
13 GBC13 Round Small Regular Flat Sticky White Smooth Opaque No GPR Singular
14 GBC14 Round Small Regular Raised Sticky White Smooth Translucent No GPR Singular, Chain
15 GBC15 Round Medium Regular Undulate Moist White Slimy Opaque No GNR Singular, Chain
16 GBC16 Round Small Irregular Flat Moist Creamy Smooth Translucent No GPR Singular, Chain
17 GBC17 Round Large Irregular Flat Moist Creamy glistening Opaque No GNR Chain
18 GBC18 Round Small Irregular Convex Dry White Smooth Opaque No GPR Singular
19 GBC19 Round Small Regular Flat Sticky White Smooth Opaque No GPR Singular
20 GBC20 Round Small Regular Raised Sticky White Smooth Translucent No GPR Singular, Chain
21 GBC21 Round Medium Regular Undulate Moist White Slimy Opaque No GNR Singular, Chain
22 GBC22 Round Small Regular Raised Moist Creamy Slimy Opaque No GNR Singular
23 GBC23 Round Large Irregular Flat Moist White glistening Translucent No GPR Singular, Chain
24 GBC24 Round Small Regular Raised Sticky White Smooth Opaque No GNR Singular, Chain
25 GBC25 Round Large Rhizoid Convex Dry White Smooth Translucent No GPR Singular
26 GBC26 Round Small Regular Raised Moist White Smooth Opaque No GNR Singular
27 GBC27 Round Small Rhizoid Convex Moist Yellow Smooth Opaque No GPR Singular, Chain

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Table 6.5. Colonial and morphological characteristics of selected microorganism from the Dragon fruits -based cropping system

Sl. Culture shape size Margin Elevation Consistency Color Surface Opacity Odour Gram’s Arrangement
No. name reaction
1 DBC1 Round Large Rhizoid Flat Moist Off-white Smooth Translucent No GNR Singular, Chain
2 DBC2 Round Medium Irregular Flat Creamy White glistening Translucent No GNR Singular, Chain
3 DBC3 Round Small Irregular Raised Moist Creamy Smooth Opaque No GPR Singular
4 DBC4 Round Small Irregular Raised Moist Creamy Smooth Opaque No GNR Singular
5 DBC5 Round Small Irregular Flat Creamy Yellow Smooth Translucent No GPR Singular, Chain
6 DBC6 Round Small Regular Raised Sticky White glistening Opaque No GNR Singular, Chain
7 DBC7 Round Medium Rhizoid Convex Moist Yellow Smooth Opaque No GPR Singular, Chain
8 DBC8 Round Medium Regular Flat Creamy White Slimy Opaque No GNR Singular, Chain
9 DBC9 Round Small Regular Flat Moist Creamy Smooth Opaque No GNR Singular
10 DBC10 Round Large Rhizoid Raised Dry White Smooth Translucent No GNR Singular
11 DBC11 Round Small Rhizoid Flat Moist White Smooth Opaque No GNR Singular, Chain
12 DBC12 Round Pinpoint Irregular Undulate Moist Creamy Smooth Translucent No GNR Singular
13 DBC13 Round Pinpoint Regular Flat Sticky White Slimy Translucent No GPR Singular
14 DBC14 Round Medium Entire Raised Moist Off-white glistening Opaque No GPR Singular, Chain
15 DBC15 Round Big Entire Raised Moist White Smooth Opaque No GPR Singular, Chain
16 DBC16 Round Medium Entire Convex Moist White Smooth Opaque No GPR Singular, Chain
17 DBC17 Round Medium Rhizoid Convex Moist Yellow Smooth Opaque No GPR Singular, Chain
18 DBC18 Round Medium Regular Flat Creamy White Slimy Opaque No GNR Singular, Chain
19 DBC19 Round Small Regular Flat Moist Creamy Smooth Opaque No GNR Singular
20 DBC20 Round Pinpoint Irregular Undulate Moist Creamy Smooth Translucent No GNR Singular, Chain
21 DBC21 Round Pinpoint Regular Flat Sticky White Slimy Translucent No GPR Singular
22 DBC22 Round Medium Entire Raised Moist Off-white glistening Opaque No GPR Singular, Chain
23 DBC23 Round Small Irregular Raised Moist Creamy Smooth Opaque No GPR Singular
24 DBC24 Round Small Irregular Raised Moist Creamy Smooth Opaque No GNR Singular
25 DBC25 Round Small Irregular Flat Creamy Yellow Smooth Translucent No GPR Singular, Chain

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Potential PGPR Bacterial Strain of Banana Based Cropping System

Potential PGPR Bacterial Strain of Citrus Based Cropping System

Potential PGPR Bacterial Strain of Guava Based Cropping System

Potential PGPR Bacterial Strain of Dragon fruits Based Cropping System

Figure 6.1.Plant Growth-Promoting Rhizobacteria (PGPR) are isolated from different agricultural
settings using selective medium.

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Banana Based Cropping System

Citrus Based Cropping System

Guava Based Cropping System

Dragon fruits Based Cropping System

Figure 6.2. Gram staining procedure distinguishes bacteria: Gram-positive (purple) have thick
peptidoglycan; Gram-negative (pink) have thin peptidoglycan with outer membrane.

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Table 6.6. Plant growth promoting activities of isolated rhizobacterial strain of banana-based cropping system. In this table plus and minus sign denote the presence and
absence PGPR activities. (+) Luxuriant Growth (O.D. ≥ 0.5), (-) Scanty/No Growth (O.D. < 0.5)

Sl No Isolate IAA Phosphate HCN Siderophore Ammonia pH Salinity Temperature

name production solubilization production production production Ph5 Ph 8 Ph 9 1% 3% 5% 4°C 25°C 42°C 55°C
1 BBC1 ++ + - - ++ - + - + + - - + + -
2 BBC 2 +++ ++ + - + + + - + - - - + + -
3 BBC 3 ++ ++ + + ++ - - - + - - - + + +
4 BBC 4 + +++ ++ - + - - + + - - - + - -
5 BBC 5 - + + + + + - + + - + - + + +
6 BBC 6 + ++ + + + + + - + + - - + + -
7 BBC 7 +++ ++ + - ++ - + + + + - + + + +
8 BBC 8 + - - - + + - + + - - - + + -
9 BBC 9 ++ +++ +++ + ++ + - - + - - - + + -
10 BBC 10 +++ +++ ++ + +++ + - - + + - - + + -
11 BBC 11 + ++ ++ - + - + + + - - - + + -
12 BBC 12 ++ + + - - + + - + + + - + + -
13 BBC 13 ++ +++ + + ++ + + - + - - + + + -
14 BBC 14 ++ ++ ++ - ++ - + + + + + - + + +
15 BBC 15 +++ +++ ++ - - + + - - - - - + - +
16 BBC 16 ++ - + - + - + - + + + - + + -
17 BBC 17 - ++ - + - - + - + + + + + + -
18 BBC 18 +++ +++ ++ + ++ - + - - - - - + - -
19 BBC 19 - ++ + - - + + - + + + - + + -
20 BBC 20 ++ +++ + - ++ + + + + - - - + - -
21 BBC 21 + ++ ++ - + - + + + - - - + + -
22 BBC 22 ++ + + - + + + - + + + - + + -
23 BBC 23 ++ +++ + + ++ + + - + - - + + + -
24 BBC 24 ++ ++ + + ++ - - - + - - - + + +
25 BBC 25 + +++ ++ - + - - + + - - - + - -
26 BBC 26 - + + + + + - + + - + - + + +
27 BBC 27 + +++ ++ - + - - + + - - - + - -
28 BBC 28 - + + + + + - + + - + - + + +
29 BBC 29 + ++ + + + + + - + + - - + + -

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Table 6.7. Plant growth promoting activities of selected microorganism of citrus-based cropping system. In this table plus and minus sign denote the presence and absence
PGPR activities. (+) Luxuriant Growth (O.D. ≥ 0.5), (-) Scanty/No Growth (O.D. < 0.5)

Serial Isolate IAA Phosphate HCN Siderophore Ammonia pH Salinity Temperature

No name production solubilization production production production Ph5 Ph8 Ph9 1% 3% 5% 4°C 25°C 42°C 55°C
1 CBC1 +++ + + + + - + - + + - + + + -
2 CBC2 ++ +++ + - + + + - + - - - + +
3 CBC3 +++ + ++ + ++ - + - + + + - + + +
4 CBC4 + +++ ++ - + + + - + - - - + - -
5 CBC5 - +++ + - + - + + + - - + + + +
6 CBC6 +++ - + + + + + + + - - - + +
7 CBC7 +++ +++ + ++ + + + - - - - + + - +
8 CBC8 ++ + - - + - + + + + + + + + -
9 CBC9 + + + + ++ + - - + - - - + + -
10 CBC10 - +++ ++ + + + + - + - - - + + -
11 CBC11 ++ ++ + - + - + + + + - + + - -
12 CBC12 +++ + + + + + - - + - - - + + -
13 CBC13 +++ ++ + + ++ - + - + + - - + - -
14 CBC14 - + ++ - ++ - + - + + - - + + +
15 CBC15 + ++ + + - + + - + + - + + + -
16 CBC16 ++ + - - + - + + + + + + + + -
17 CBC17 + + + - + + - - + - - - + + -
18 CBC18 - ++ ++ + + + + - + - - - + + -
19 CBC19 ++ + - - + - + + + + + + + + -
20 CBC20 + + + + ++ + - - + - - - + + -
21 CBC21 - +++ ++ + + + + - + - - - + + -
22 CBC22 - +++ + + + - + + + - - + + + +
23 CBC23 ++ - + - + + + - + - - - + +
24 CBC24 +++ ++ + ++ + + + - - - - + + - +
25 CBC25 + + - - + - + + + + + - + + -
26 CBC26 - ++ + - + - + + + - - + + + +

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Table 6.8. Plant growth promoting activities of selected microorganism of Guava-based cropping system. In this table plus and minus sign denote the presence and absence
PGPR activities. (+) Luxuriant Growth (O.D. ≥ 0.5), (-) Scanty/No Growth (O.D. < 0.5)

Sl Isolate IAA Phosphate HCN Siderophore Ammonia pH Salinity Temperature

No name production solubilization production production production Ph5 Ph Ph 1% 3% 5% 4°C 25°C 42°C 55°C
8 9
1 GBC1 +++ +++ + + ++ + + - + + - + + + -
2 GBC2 ++ +++ + - + + + - + - - - + + -
3 GBC3 +++ ++ + + ++ - + + - - - - + + +
4 GBC4 + +++ ++ - + + - - + + - - + + -
5 GBC5 ++ + + + + - + + + - - + + + +
6 GBC6 ++ - + + + + + + + - - - + + -
7 GBC7 - +++ + ++ + + - - - - - - + - -
8 GBC8 + +++ - - + - + + + + + + + + +
9 GBC9 ++ + + + ++ + - - + - - - + + -
10 GBC10 - ++ + - + + + - + - - - + + +
11 GBC11 ++ +++ - + + - + + + + - + + - +
12 GBC12 +++ + + + + + - - + + + - + + +
13 GBC13 + ++ - + ++ - + - + + - - + - -
14 GBC14 +++ + + - ++ - + - + + + - + + +
15 GBC15 - ++ + + - + - - + + - + + + -
16 GBC16 ++ + + + + + - - - - - - + + -
17 GBC17 + + + - + - + - + - - - + - -
18 GBC18 + - - + + - - - - - - - + - -
19 GBC19 + ++ - - ++ - + - + + - - + - -
20 GBC20 ++ + + - ++ - + - + + + - + + +
21 GBC21 - ++ + + - + + - + + - + + + +
22 GBC22 - + + ++ + + - - - - - - + - -
23 GBC23 + ++ - - + - + + + + + + + + +
24 GBC24 ++ + - + ++ - - - + - - - + + -
25 GBC25 - ++ + - + + - - + - - - + -
26 GBC26 + ++ - - + - + + + + + + + + +
27 GBC27 ++ + + + ++ + - - + - - - + + -

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Table 6.9. Plant growth promoting activities of selected microorganism of Dragon fruits-based cropping system. In this table plus and minus sign denote the presence and
absence PGPR activities. (+) Luxuriant Growth (O.D. ≥ 0.5), (-) Scanty/No Growth (O.D. < 0.5)

Serial Isolate IAA Phosphate HCN Siderophore Ammonia pH Salinity Temperature

No name production solubilization production production production Ph5 Ph8 Ph9 1% 3% 5% 4°C 25°C 42°C 55°C
1 DBC1 + + + + + - - - + - - - + + -
2 DBC2 ++ + + - + + - + - - - + - -
3 DBC3 ++ ++ + + + - + + - - - - + - -
4 DBC4 + ++ ++ - - + - - + - - - + + -
5 DBC5 +++ + + + + - + + + + - - + + +
6 DBC6 ++ +++ + - + + + - + - - - + + -
7 DBC7 +++ +++ + + + - + + + + - - + - -
8 DBC8 - ++ - - + - + - + + - + + - -
9 DBC9 ++ + + + ++ - - - + - - - + + -
10 DBC10 - +++ + + + + + - + - - - + + -
11 DBC11 +++ - - + + - + + + + - + + - -
12 DBC12 ++ ++ ++ + + + - - - - - - + + -
13 DBC13 + ++ - + + - + - - - - - + - -
14 DBC14 +++ + + - ++ - + - + + + - + + +
15 DBC15 - +++ + + - + + + + + - + + + -
16 DBC16 ++ ++ + - + + - - - - - - + + -
17 DBC17 - ++ - + + - + - + + - + + - -
18 DBC18 ++ + + + ++ - - - + - - - + + -
19 DBC19 - +++ + + + + + - + - - - + + -
20 DBC20 ++ + + - ++ - + - + + + - + + +
21 DBC21 - ++ + - - + + + + + - + + + -
22 DBC22 ++ ++ + + + + - - - - - - + + -
23 DBC23 - ++ - - + - + - + + - + + - +
24 DBC24 - ++ - - + - + + + + - + + - -
25 DBC25 ++ + + + ++ - - - + - - - + + +

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6.3.4. Salinity sensitive test of isolated rhizobacterial strain of diversified cropping

system
Salinity-sensitive rhizobacteria play an important role in sustainable agriculture by
helping plants withstand salt stress (Ditta et al., 2023; Qadir et al. 2008; Lehman et al.
2015). Tables 6.6 to 6.9 demonstrate their adaptability to various cropping patterns.
Various banana strains grow at salt levels of 1%, 3%, and 5%. Similarly, in citrus, guava,
and dragon fruit systems, these bacteria exhibit varying salinity-induced growth
responses. Citrus farming, for example, thrives at 1% and 3% salinity, whereas guava
production excels at 1% and 3% salinity, with fewer strains surviving at 5% salinity.
These findings highlight rhizobacteria's ability to improve crop resilience in adverse
conditions, hence contributing to sustainable agriculture methods.
6.3.5. Temperature sensitive test of isolated rhizobacterial strain of diversified
cropping system
Temperature-sensitive rhizobacteria are critical to crop variety and adaptation (Ashraf et
al. 2019; Dubey et al. 2019; Beddington et al. 2011). The adaptability of banana, citrus,
guava, and dragon fruit systems varies with temperature (40°C, 25°C, 42°C, and 55°C.
Table 6.6 demonstrates that only four strains grow at 40°C, while the majority flourish at
25°C and 42°C. Interestingly, 8 strains can endure 55°C. Similarly, strains in the citrus
(Table 6.7) and guava (Table 6.8) systems grow at varying temperatures, with some
surviving at 55°C. Dragon fruit cropping (Table 6.9) displays strains that grow at 40°C
and 55°C, with optimal growth at 25°C and 42°C. Understanding these adaptabilities is
critical for resilient agriculture in many climates.

6.3.6. Plant growth hormones production from isolated rhizobacterial strain of

diversified cropping system
In diversified cropping systems, isolated rhizobacterial strains produce plant growth
hormones that enhance nutrient uptake, solubilize minerals, fix nitrogen, and boost plant
health. These bacteria adapt to diverse root exudates, promoting growth, resilience, and
sustainability within the agricultural ecosystem. They stimulate root growth, nutrient
uptake, stress tolerance, and establish symbiotic relationships with different crops,
enhancing nutrient solubilization, nitrogen fixation, and overall plant health. This
interplay results in an optimized ecosystem for plant flourishing.

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6.3.6.1.IAA production
Bacteria that produce indole-3-acetic acid (IAA) are essential for varied cropping systems
because they enhance plant vigor, vigorous development, and nutrient absorption. They
support microbial activity, preserve soil health, and assist plants in fending off
environmental challenges. Bacteria that produce IAA help plants become more adaptable,
which encourages sustainable farming methods and resilience in a variety of crop
situations (Hakim et al. 2021). The PGPR isolates from the banana-based cropping
system were assessed for their in vitro capacity to produce indole acetic acid (IAA). The
findings are shown in Table 6.6. and Figure 6.3 Out of the twenty-nine bacterial isolates
used in the banana-based cropping system, 25 were found to produce indole acetic acid.
The isolates BBC 2, BBC 7, BBC 10, BBC 15, and BBC 18 generated the most IAA. On
the other hand, 16 bacterial strains out of 26 were discovered to produce IAA in the
citrus-based cropping system. Table 6.7. displays the highest quantity of IAA production
in the CBC 1, CBC 3, CBC6, CBC7, CBC12, CBC13, and CBC24. Additionally, Table
6.8 depicts the cropping system based on guavas. The results indicate that, of the twenty-
seven bacterial strains tested, 21 of them produced IAA. The GBC1, GBC3, GBC12, and
GBC 14 had the highest levels of IAA production (Table 6.8.). On the other hand, 17
rhizobacterial isolates in the cropping system based on dragon fruits demonstrate the
successful generation of IAA. Bacterial strains DBC5, DBC7, DBC11, and DBC14 have
the highest levels of IAA generation (Table 6.9.).

6.3.6.2. Phosphate solubilization

PSB (phosphorus-solubilizing bacteria) are important in diverse cropping systems
because they increase phosphorus availability for plants. They create enzymes and acids
that break down insoluble phosphorus, facilitating nutrient intake and encouraging
vigorous development. PSB aid in nitrogen cycling, lowering dependency on synthetic
fertilizers, and increasing agricultural production in a sustainable manner (Bargaz et al.
2021). The ability of the PGPR isolates from the banana-based cropping system to
produce Phosphate solubilization (PSB) was evaluated in vitro. The results are presented
in Table 6.6. and Figure 6.3. Among the twenty-nine bacterial isolates employed in the
banana-based cropping system, a total of 25 were shown to possess the ability to convert
insoluble mineral phosphate into a soluble form. The rhizobacterial isolates BBC 4, BBC

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9, BBC 10, BBC 13, BBC 15, BBC 18, BBC 20, BBC 23, BBC 25, and BBC 27 exhibit
the ability to dissolve a significant amount of insoluble mineral phosphate. In contrast,
the citrus-based cropping system exhibited the largest amount of solubilized insoluble
mineral phosphate in CBC 2, CBC 4, CBC 5, CBC 7, CBC 10, CBC 21, and CBC 22
(Table 6.7.). Furthermore, Table 6.8. presents the guava-based cropping system. The
findings demonstrate that bacteria strains GBC1, GBC2, GBC4, GBC7, GBC8, and
GBC11 exhibited the most significant capacity to dissolve mineral phosphate, as
indicated in Table 6.8. Rhizobacterial isolates in the dragon fruit farming system
effectively solubilize phosphate minerals. The bacterial strains DBC6, DBC7, DBC10,
DBC15, and DBC19 have the greatest capacity for solubilizing mineral phosphate.

6.3.6.3. HCN Production

Hydrogen cyanide (HCN)-producing rhizobacterial strains improve soil health, naturally
suppress pests and diseases, and promote plant development, all of which help to
agricultural diversity. By fostering vigorous plant development, a better soil environment,
and sustainable combating pests, this strategy reduces the need for synthetic chemicals in
agriculture (Khatoon et al. 2020). The in vitro evaluation was conducted to assess the
HCN generation potential of the PGPR isolates obtained from the banana-based cropping
system. The findings are displayed in Table 6.6. and Figure 6.3. Out of the twenty-nine
bacterial isolates used in the banana-based cropping system, a total of 16 were shown to
have the capability to produce hydrogen cyanide (HCN). The rhizobacterial isolates BBC
9, BBC 10, BBC 11, BBC 14, BBC 15, and BBC 27 have the capability to produce
hydrogen cyanide (HCN). The citrus-based cropping system showed the highest levels of
hydrogen cyanide (HCN) generation in CBC 3, CBC 4, CBC 10, CBC 14, CBC 18, and
CBC 21, as indicated in Table 6.7. Additionally, Table 6.8. displays the cropping
mechanism that is based on guava. The results reveal that the bacteria strains GBC4
showed the highest ability to produce HCN, as shown in Table 6.8. However,
Rhizobacterial isolates in the dragon fruit farming system exhibit efficient hydrogen
cyanide (HCN) production. The bacterial strains DBC4 and DBC12 exhibit the highest
capability for hydrogen cyanide (HCN) production.

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6.3.6.4. Siderophore production

Rhizobacterial strains produce siderophores, which facilitate the absorption of iron and
enhance nutrient accessibility in several agricultural systems. The formation of these
compounds results in the creation of soluble complexes, which in turn promotes plant
development and decreases the likelihood of disease susceptibility (Sayyed et al.2012).
Additionally, they surpass pathogens in the competition for iron, therefore enhancing the
overall health of plants and maintaining a harmonious soil microbial environment, which
in turn supports the implementation of sustainable agricultural methods (Meena et al.
2017). The ability of the PGPR isolates from the banana-based cropping system to
produced siderophore was evaluated in vitro. The results are presented in Table 6.6. and
Figure 6.3. Among the twenty-nine bacterial isolates employed in the banana-based
cropping system, a total of 13 were seen to exhibit siderophore production. In contrast, 14
out of 26 bacterial strains were shown to generate siderophore in the citrus-based
cropping system (Table 6.7.). Furthermore, Table 6.8. presents the guava-based cropping
system. The findings demonstrate that out of the twenty-seven bacterial strains examined,
13 of them exhibited siderophore production. However, in the cropping system focused
on dragon fruits, 15 rhizobacterial isolates have shown effective production of
siderophore.

6.3.6.5. Ammonia Production

Rhizobacterial ammonia-producing strains are essential in many cropping systems
because they transform organic nitrogen into plant-absorbable ammonia, which is
required for development (Chaudhary et al. 2019). This technique improves crop health
by influencing soil pH, optimizing nutrient availability, and suppressing soil borne
pathogens (Panth yet al. 2020). Rhizobacterial strains also contribute to nitrogen cycling,
which reduces dependency on synthetic fertilizers and promotes sustainable agriculture.
They maintain nutrient availability, control pH, inhibit pathogens, and manage nitrogen in
a sustainable manner, enabling robust plant development in a variety of cropping settings
(Hamid, et al. 2021). The PGPR isolates from the banana-based cropping system were
assessed for their in vitro capacity to produce ammonia.

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The findings are shown in Table 6.6 and Figure 6.3 The isolates from banana-based
cropping system BBC 3, BBC 7, BBC 9, BBC 10, BBC 13, BBC 14 and BBC 18
generated the most ammonia. On the other hand, citrus based cropping system, Table 6.7
displays the highest quantity of ammonia production in the CBC 3, CBC9, CBC13,
CBC14, and CBC20. Additionally, Table 6.8. displays the cropping system based on
guavas. The results indicate that bacterial strain such as GBC1, GBC3, GBC9, GBC13,
GBC14, GBC19and GBC 20 had the highest levels of ammonia production (Table 6.8.).
On the other hand, rhizobacterial isolates in the dragon fruits-based cropping system
based such as DBC 9, DBC14, DBC,18, DBC 20 and DBC 25 produced ammonia.

6.3.7. Mineral bio-dissolution by PGPR isolates of diversified cropping system

6.3.7.1. Bio-dissolution of insoluble phosphates and release of phosphorus

The isolates' ability to break down insoluble phosphates and release phosphorus (P) was
evaluated using both qualitative and quantitative approaches. The qualitative examination
entailed examining changes in phosphate compounds, whilst the quantitative evaluation
examined the amount of phosphorus released, determining the phosphorus solubilization
capability of the isolates. Both approaches were used to assess the isolates' ability to
transform unavailable phosphorus into a form that plants can use.

6.3.7.2. Plate assay

The study evaluated PGPR isolates' ability to solubilize insoluble mineral phosphate in
different cropping systems (Deepa et al. 2015) determining the Phosphate Solubilizing
Activity Index (PSI) at 3, 5, and 7 days after inoculation (DAI). For the banana-based
system (Table 6.10), PSI values ranged from 3.02 to 4.06, with BBC 18 exhibiting the
highest PSI at 3DAI (3.57). In the citrus system (Table 6.11), PSI ranged from 3.00 to
4.20, with CBC 5 showing the highest PSI at 5DAI (4.18). For the guava system (Table
6.12), PSI ranged from 2.98 to 3.62, with GBC 11 having the highest PSI at 3DAI (3.44).
In the dragon fruit system (Table 6.13), PSI ranged from 2.96 to 3.60, with DBC 10
showing the highest PSI at 5DAI (3.49). These results demonstrate the varying phosphate
solubilization capabilities of rhizobacterial strains across different crops and time points.

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Test for PGPR activity

Potassium Solubilization Phosphate Solubilization

Siderophore Production Zinc solubilization

HCN Production Ammonia Production

Indole-3-acetic acid (IAA) Production

Figure 6.3. Assessing PGPR entails evaluating plant growth, phosphate solubilization, nitrogen
fixation, hormone synthesis, and pathogen defense, enhancing sustainable agriculture and soil
fertility.

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Table 6.10. Qualitative estimation of phosphate solubilization of five promising bacteria of banana-based
cropping system on Pikovskaya agarmedium at 3 DAI, 5DAI, 7DAI

Phosphate solubilization
Bacteria 3DAI 5DAI 7DAI
isolate P halo zone Colony PSI P halo zone Colony PSI P halo Colony PSI
(mm) diameter (mm) diameter zone diameter
(mm) (mm) (mm) (mm)
BBC1 2.65±0.22 2.23±0.08 2.18±0.01 4.21±0.12 5.45±0.04 1.77±0.02 5.12±0.12 6.21±0.08 1.82±0.01
BBC2 5.26±0.11 4.56±0.05 2.15±0.05 7.24±0.25 6.45±0.25 2.12±0.03 7.28±0.13 6.74±0.14 2.08±0.02
BBC3 3.25±0.15 4.36±0.01 1.74±0.06 5.63±0.31 4.58±0.14 2.23±0.04 5.86±0.25 4.62±0.16 2.27±0.05
BBC4 10.5±0.21 5.26±0.05 3.02±0.01 14.25±0.14 6.56±0.13 3.17±0.05 14.8±0.14 6.85±0.31 3.17±0.04
BBC8 6.58±0.14 3.25±0.06 1.49±0.36 8.56±0.36 6.45±0.16 2.32±0.01 8.7±0.32 6.56±0.85 2.33±0.06
BBC9 4.56±0.13 4.26±0.01 2.07±0.21 6.25±0.01 5.40±0.18 2.15±0.01 6.85±0.14 5.87±0.12 2.17±0.04
BBC7 7.23±0.25 4.25±0.15 2.70±0.05 8.15±0.05 6.25±0.13 2.30±0.03 8.16±0.13 6.36±0.14 2.28±0.11
BBC8 2.26±0.14 3.25±0.05 1.69±0.05 3.85±0.42 4.25±0.16 1.90±0.05 4.2±0.15 4.52±0.14 1.93±0.04
BBC9 16.2±0.11 6.45±0.06 3.51±0.14 18.26±0.05 6.25±0.11 3.92±0.05 19.2±0.02 6.35±0.36 4.03±0.01
BBC10 3.56±0.13 5.58±0.08 1.63±0.25 5.26±0.10 3.45±0.13 2.52±0.04 5.35±0.05 4.15±0.75 2.29±0.03
BBC11 12.3±0.21 4.86±0.04 3.54±0.36 14.26±0.02 5.15±0.23 3.76±0.12 16.2±0.08 5.48±0.12 3.97±0.05
BBC12 8.26±0.25 5.25±0.02 2.57±0.02 10.2±0.06 5.46±0.25 2.86±0.21 10.5±0.75 5.96±0.18 2.76±0.02
2BBC13 2.25±0.31 2.26±0.32 1.99±0.05 5.24±0.04 6.12±0.36 1.85±0.05 7.25±0.08 6.34±0.13 2.14±0.04
BBC14 4.26±0.14 3.45±0.14 2.23±0.01 6.24±0.02 4.25±0.12 2.46±0.15 6.85±0.11 4.60±0.05 2.49±0.06
BBC15 10.56±0.12 4.16±0.15 3.53±0.36 12.3±0.07 4.18±0.02 3.95±0.06 13.2±0.04 4.85±0.16 3.73±0.05
BBC16 3.25±0.25 4.63±0.63 1.70±0.15 4.56±0.05 4.25±0.52 2.07±0.08 5.21±0.13 4.65±0.41 2.12±0.04
BBC17 4.36±0.31 4.15±0.85 2.05±0.24 5.56±0.32 3.56±0.14 2.56±0.31 6.21±0.07 4.2±0.03 2.48±0.14
BBC18 13.25±0.01 5.15±0.04 3.57±0.36 16.2±0.04 5.48±0.12 3.96±0.04 17.2±0.08 5.64±0.02 4.06±0.05
BBC19 3.56±0.05 3.85±0.05 1.92±0.14 5.26±0.12 4.25±0.4 2.23±0.02 6.21±0.07 4.87±0.05 2.28±0.12
BBC20 8.5±0.08 5.19±0.31 2.63±0.25 9.45±0.11 4.62±0.23 3.04±0.36 9.82±0.12 4.98±0.15 2.97±0.11

Table 6.11. Qualitative estimation of phosphate solubilization of five promising bacteria of Citrus-based
cropping system on Pikovskaya agar medium at 3 DAI, 5DAI, 7DAI

Phosphate solubilization
Bacteria 3DAI 5DAI 7DAI
isolate
P halo Colony PSI P halo Colony PSI P halo Colony PSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
CBC1 3.21± 0.25 2.25±0.25 2.43±0.45 4.12±0.25 3.25±0.16 2.27±0.85 4.28±0.75 4.25±0.31 2.01±0.12
CBC2 2.45±0.58 3.14±0.85 1.78±0.15 3.25±0.14 3.85±0.15 1.84±0.45 3.85±0.45 5.21±0.32 1.74±0.11
CBC3 4.25±0.56 2.15±0.42 2.98±0.26 5.12±0.14 3.15±0.32 2.63±0.24 6.25±0.12 4.85±0.35 2.29±0.08
CBC4 9.12±0.14 4.56±0.26 3.00±0.25 10.52±0.25 4.95±0.14 3.13±0.16 12.35±0.12 5.24±0.62 3.36±0.08
CBC5 12.25±0.75 4.56±0.14 3.68±0.25 15.25±0.16 4.8±0.78 4.18±0.31 16.25±0.11 5.12±0.14 4.20±0.06
CBC6 5.64±0.25 4.25±0.26 2.33±0.14 6.25±0.15 5.42±0.98 2.15±0.15 7.25±0.15 6.21±0.25 2.17±0.04
CBC7 11.25±0.45 5.26±0.75 3.14±0.16 13.25±0.26 4.68±0.85 3.83±0.48 14.23±0.13 5.23±0.34 3.72±0.03
CBC8 6.25±00.48 3.85±0.85 2.62±0.14 6.85±0.16 4.26±0.56 2.61±0.64 7.25±0.14 4.89±0.15 2.48±0.04
CBC9 4.25±0.74 3.75±0.26 2.13±0.18 5.21±0.18 4.26±0.25 2.22±0.25 6.25±0.45 4.7±0.23 2.33±0.08
CBC10 10.85±0.15 5.21±0.25 3.13±0.25 12.45±0.17 5.84±0.36 3.13±0.85 13.25±0.32 6.25±0.15 3.12±0.15
CBC11 2.69±0.14 3.25±0.84 1.83±0.63 3.25±0.96 4.26±0.14 1.76±0.64 3.58±0.23 5.12±014 1.70±0.12
CBC12 3.58±0.36 2.58±0.96 2.39±0.85 4.25±0.15 3.89±0.63 2.09±0.34 4.89±0.15 4.26±0.36 2.15±0.13
CBC13 4.2±0.28 4.12±0.52 2.02±0.63 4.85±0.28 4.89±0.85 1.99±0.85 5.25±0.41 5.21±0.15 2.01±0.12
CBC14 1.55±0.25 2.31±0.74 1.71±0.45 2.56±0.26 3.25±0.56 1.79±0.74 3.18±0.13 4.23±0.31 1.73±0.14
CBC15 10.45±0.14 3.15±0.15 3.31±0.42 11.5±0.18 4.15±0.36 3.80±0.46 12.41±0.35 4.85±0.25 3.56±0.11

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Table 6.12. Qualitative estimation of phosphate solubilization of bacteria of Guava-based cropping system
on Pikovskaya agar medium at 3 DAI, 5DAI, 7DAI

Phosphate solubilization
Bacteria 3DAI 5DAI 7DAI
isolate P halo Colony PSI P halo zone Colony PSI P halo zone Colony PSI
zone (mm) diameter (mm) diameter (mm) diameter
(mm) (mm) (mm)
GBC1 12.25±0.25 5.64±0.45 3.17±0.15 14.23±0.85 6.25±0.12 3.28±0.12 15.23±0.21 6.85±0.35 3.22±0.11
GBC2 2.25±0.23 3.56±0.75 1.63±0.05 3.25±0.45 4.26±0.13 1.76±0.08 4.52±0.31 5.26±0.85 1.86±0.12
GBC3 3.56±0.56 4.25±0.46 1.84±0.06 4.25±0.96 4.85±0.32 1.88±0.09 5.26±0.15 5.24±0.87 2.00±0.12
GBC4 13.56±0.45 6.12±0.15 3.22±0.15 15.26±0.45 6.89±0.52 3.21±0.15 16.25±0.36 7.25±0.54 3.24±0.18
GBC5 4.58±0.36 3.89±0.85 2.18±0.16 4.89±0.23 4.25±0.31 2.15±0.11 5.87±0.45 5.26±0.64 2.12±0.08
GBC6 3.42±0.75 3.45±0.36 1.99±0.25 3.75±0.25 4.56±0.85 1.82±0.12 4.56±0.63 5.43±0.56 1.84±0.09
GBC7 9.89±0.85 4.58±0.23 3.16±0.36 11.25±0.12 5.26±0.12 3.14±0.13 13.52±0.74 5.98±0.15 3.26±0.06
GBC8 11.25±0.15 5.25±0.58 3.14±0.52 12.35±0.13 6.25±0.41 2.98±0.01 15.47±0.15 7.25±0.15 3.13±0.14
GBC9 6.52±0.23 4.26±0.15 2.53±0.34 7.25±0.31 5.24±0.12 2.38±0.02 8.56±0.25 6.25±0.41 2.37±0.12
GBC10 8.25±0.36 4.36±0.16 2.89±0.16 9.25±0.42 5.84±0.56 2.58±0.02 10.56±0.63 6.28±0.63 2.68±0.21
GBC11 13.85±0.75 5.29±0.45 3.62±0.12 15.26±0.31 5.89±0.31 3.59±0.13 16.52±0.15 6.78±0.15 3.44±0.22
GBC12 4.26±0.15 3.56±0.16 2.20±0.15 5.26±0.26 4.89±0.15 2.08±0.15 7.52±0.52 5.89±0.85 2.28±0.21
GBC13 5.26±0.36 4.26±0.15 2.23±0.15 5.89±0.13 5.78±0.12 2.02±0.03 6.58±0.96 6.89±0.74 1.96±0.14
GBC14 6.54±0.15 3.56±0.85 2.84±0.36 7.25±0.15 4.59±0.35 2.58±0.15 8.56±0.63 7.25±0.26 2.18±0.09
GBC15 7.25±0.85 4.25±0.36 2.71±0.15 8.25±0.13 5.21±0.25 2.58±0.04 9.25±0.85 6.78±0.12 2.36±0.07
GBC16 6.35±0.45 3.56±0.12 2.78±0.15 7.26±0.12 4.68±0.85 2.55±0.11 8.56±0.25 6.89±0.14 2.24±0.09
GBC17 4.26±0.36 3.65±0.85 2.17±0.16 5.26±0.31 4.32±0.13 2.22±0.13 6.25±0.45 7.25±0.56 1.86±0.15
GBC18 5.23±0.15 4.25±0.15 2.23±0.09 6.23±0.52 5.28±0.15 2.18±0.12 7.26±0.31 7.56±0.42 1.96±0.13

Table 6.13. Qualitative estimation of phosphate solubilization of five promising bacteria of Dragon fruits-
based cropping system on Pikovskaya agar medium at 3 DAI, 5DAI, 7DAI
Phosphate solubilization
Bacteria 3DAI 5DAI 7DAI
isolate P halo Colony PSI P halo Colony PSI P halo Colony PSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
DBC1 3.25±0.25 3.25±0.52 2.00±0.15 4.42±0.09 4.25±0.15 2.04±0.23 5.52±0.45 5.42±0.11 2.02±0.31
DBC2 5.26±0.15 4.25±0.63 2.24±0.63 6.23±0.06 5.26±0.52 2.18±0.15 6.89±0.75 5.89±0.23 2.17±0.11
DBC3 6.56±0.45 4.26±0.14 2.54±0.12 7.25±0.01 5.46±0.15 2.33±0.14 8.15±0.45 6.25±0.12 2.30±0.10
DBC4 3.25±0.78 3.85±0.34 1.84±0.14 4.26±0.05 4.85±032 1.88±0.08 6.25±0.36 5.24±0.14 2.19±0.08
DBC5 13.25±0.15 5.63±0.52 3.35±0.36 15.26±0.06 6.25±0.18 3.44±0.09 17.25±0.23 7.25±0.13 3.38±0.07
DBC6 15.23±0.15 6.23±0.45 3.44±0.56 17.21±0.12 7.28±0.45 3.36±0.08 18.25±0.15 7.84±0.32 3.33±0.06
DBC7 12.45±0.16 5.49±0.32 3.27±0.15 13.25±0.13 6.45±0.16 3.05±0.15 14.26±0.85 7.26±0.52 2.96±0.12
DBC8 4.52±0.25 4.25±0.45 2.06±0.14 5.26±0.11 5.26±0.15 2.00±0.14 6.23±0.12 6.25±0.14 2.00±0.14
DBC9 6.32±0.45 3.56±0.74 2.78±0.15 7.26±0.14 4.85±0.32 2.50±0.14 8.25±0.15 5.63±0.11 2.47±0.14
DBC10 10.52±0.52 5.28±0.85 2.99±0.12 12.35±0.08 4.96±0.52 3.49±0.01 14.26±0.36 5.48±0.31 3.60±0.31
DBC11 4.59±0.36 3.21±0.32 2.43±0.36 5.26±0.05 4.86±0.25 2.08±0.02 6.25±0.45 5.96±0.14 2.05±0.14
DBC12 10.85±0.25 4.85±0.11 3.24±0.45 12.35±0.12 5.26±0.14 3.35±0.03 14.23±0.45 6.35±0.25 3.24±0.12
DBC13 2.36±0.45 3.25±0.13 1.73±0.12 3.25±0.13 4.65±0.85 1.70±0.52 4.23±0.32 5.48±0.85 1.77±0.15
DBC14 3.25±0.36 4.21±0.36 1.77±0.15 4.26±0.15 5.85±0.36 1.73±0.15 5.23±0.15 6.25±0.36 1.84±0.12
DBC15 4.26±0.85 3.56±0.15 2.20±0.36 5.26±0.16 4.85±0.14 2.08±0.14 6.23±0.12 5.84±0.14 2.07±0.08
DBC16 3.25±0.41 3.45±0.36 1.94±0.15 4.26±0.14 6.8±0.25 1.63±0.31 5.26±0.31 7.25±0.14 1.73±0.13

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6.3.8. Bio-dissolution of insoluble silicates and release of potassium and silica

The microbial-mediated process of bio-dissolving insoluble silicates, such as through

the activity of potassium solubilizing bacteria (KSB), plays a vital role in agriculture
by rendering potassium accessible to plants from previously inaccessible sources,
such as insoluble silicates present in the soil (Raj et al., 2010). This procedure
improves soil fertility and promotes the ability of plants to endure various
pressures.The investigation of these bacteria's capacity to extract potassium from
insoluble sources is yielding vital insights for the development of sustainable farming
techniques. Aleksandrow agar, a specific medium for cultivation, can be employed to
investigate the bio-dissolution of insoluble silicates by isolated bacteria.Through the
examination of the constituents of the medium and the growth patterns of bacteria,
scientists can get a deeper comprehension of the mechanisms underlying the
liberation of potassium and silica from these sources that are difficult to reach (Sood
et al. 2023).

6.3.8.1. Plate assay

The research delved into the role of Plant Growth-Promoting Rhizobacteria (PGPR)
in enhancing potassium availability, crucial for optimal plant growth ‘(Bahadur et al.
2017). Key bacterial isolates exhibited significant potassium solubilization activity
across various cropping systems. For instance, strains BBC 18, CBC 13, GBC 10, and
DBC 9 demonstrated notable efficacy in solubilizing potassium in banana, citrus,
guava, and dragon fruit systems, respectively. The potassium solubilization activity
index (KSI) values ranged between 2.95 to 4.78, 3.32 to 4.39, 3.03 to 4.09, and 3.27
to 4.36 across different systems at various time points (Tables 6.14 to 6.17). These
findings underscore the potential of specific PGPR strains to improve potassium
availability, thereby contributing to enhanced plant growth and agricultural
productivity in diverse cropping environments.

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Table 6.14. Qualitative estimation of Potassium solubilization by selected isolates from Banana based
cropping system on Aleksandrov agar medium at 3 DAI, 5DAI, 7DAI

Potassium solubilization
Bacteria 3DAI 5DAI 7DAI
isolate K halo Colony KSI K halo Colony KSI K halo Colony KSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
BBC1 4.26±0.25 4.26±0.21 2.00±0.08 5.23±0.15 5.26±0.31 1.99±0.06 6.25±0.42 6.23±0.41 2.00±0.11
BBC2 10.25±0.35 5.26±0.15 2.95±0.08 13.25±0.14 6.23±0.52 3.13±0.14 14.26±0.63 6.45±0.52 3.21±0.09
BBC3 3.56±0.45 3.25±0.34 2.10±0.06 5.26±0.78 4.25±0.42 2.24±0.63 6.23±0.54 5.28±0.31 2.18±0.07
BBC4 8.26±0.14 6.24±0.36 2.32±0.07 9.56±0.96 6.89±0.63 2.39±0.14 10.26±0.63 7.25±0.15 2.42±0.08
BBC5 5.26±0.16 4.26±0.15 2.23±0.05 6.58±0.45 5.28±0.74 2.25±0.04 7.21±0.24 6.25±0.31 2.15±0.06
BBC6 6.34±0.14 4.58±0.14 2.38±0.15 7.26±0.63 5.96±0.12 2.22±0.03 7.62±0.12 6.32±0.52 2.21±0.14
BBC7 8.26±0.16 5.35±0.35 2.54±0.16 9.25±0.45 6.23±0.16 2.48±0.01 10.25±0.34 7.25±0.12 2.41±0.11
BBC8 12.35±0.11 4.29±0.85 3.88±0.14 14.63±0.15 5.78±0.12 3.53±0.05 16.23±0.15 6.25±0.11 3.60±0.01
BBC9 2.36±0.41 3.25±0.16 1.73±0.74 3.56±0.14 6.25±0.15 1.57±0.15 4.23±0.14 7.23±0.63 1.59±0.26
BBC10 6.24±0.63 6.25±0.11 2.00±0.16 7.26±0.75 6.78±0.36 2.07±0.42 8.32±0.11 7.21±0.15 2.15±0.31
BBC11 4.36±0.78 4.26±0.15 2.02±0.11 5.96±0.16 4.89±0.18 2.22±0.31 6.85±0.15 5.26±0.74 2.30±0.05
BBC12 5.12±0.16 5.28±0.36 1.97±0.01 6.89±0.35 6.87±0.34 2.00±0.74 7.25±0.34 7.32±0.52 1.99±0.14
BBC13 13.25±0.85 4.63±0.14 3.86±0.12 15.26±0.26 5.26±0.62 3.90±0.14 17.25±0.41 6.23±0.16 3.77±0.03
BBC14 3.25±0.45 5.26±0.85 1.62±0.15 4.25±0.85 6.89±0.85 1.62±0.11 5.23±0.35 7.15±0.42 1.73±0.04
BBC15 4.26±0.63 3.56±0.15 2.20±0.33 5.96±0.85 5.26±0.32 2.13±0.32 6.35±0.25 6.23±0.31 2.02±0.05
BBC16 5.26±0.33 4.26±0.36 2.23±0.14 6.35±0.24 6.25±0.16 2.02±0.24 7.26±0.74 6.89±0.25 2.05±0.06
BBC17 3.56±0.15 3.85±0.11 1.92±0.25 6.25±0.36 4.85±0.45 2.29±0.34 7.25±0.31 5.26±0.41 2.38±0.05
BBC18 14.56±0.31 3.85±0.25 4.78±0.14 16.85±0.26 4.89±0.32 4.45±0.11 17.25±0.41 6.23±0.21 3.77±0.45
BBC19 10.56±0.15 4.29±0.12 3.46±0.13 13.52±0.48 5.89±0.33 3.30±0.21 14.26±0.12 6.21±0.31 3.30±0.41
BBC20 7.59±0.22 3.78±0.47 3.01±0.18 8.56±0.15 6.25±0.85 2.37±0.31 9.25±0.42 7.15±0.56 2.29±0.63

Table 6.15. Qualitative estimation of Potassium solubilization by selected isolates from Citrus based
cropping system on Aleksandrov agar medium at 3DAI, 5DAI, 7DAI

Potassium solubilization
Bacteria 3DAI 5DAI 7DAI
isolate
K halo Colony KSI K halo Colony KSI K halo Colony KSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
CBC1 5.26±0.15 5.26±0.12 2.00±0.08 6.25±0.15 5.89±0.24 2.06±0.11 6.85±0.12 6.23±0.21 2.10±0.11
CBC2 4.26±0.41 4.85±0.41 1.88±0.04 5.26±0.24 5.21±0.25 2.01±0.15 5.89±0.45 5.89±0.45 2.00±0.12
CBC3 12.25±0.46 5.26±0.52 3.33±0.05 16.25±0.32 5.89±0.34 3.76±0.31 18.26±0.13 6.25±0.63 3.92±0.12
CBC4 5.21±0.74 4.65±0.63 2.12±0.12 6.23±0.24 5.21±0.15 2.20±0.08 6.87±0.52 5.84±0.41 2.18±0.12
CBC5 6.58±0.15 5.84±0.15 2.13±0.11 7.21±0.15 6.24±0.46 2.16±0.01 7.65±0.63 6.25±0.25 2.22±0.14
CBC6 11.26±0.35 4.85±0.42 3.32±0.13 14.23±0.34 5.12±0.32 3.78±0.18 16.32±0.15 5.89±0.74 3.77±0.13
CBC7 6.58±0.15 5.26±0.51 2.25±0.45 7.32±0.15 6.23±0.85 2.17±0.12 7.59±0.15 6.24±0.85 2.22±0.15
CBC8 7.58±0.41 4.28±0.41 2.77±0.12 8.21±0.75 5.24±0.45 2.57±0.14 8.94±0.42 5.87±0.12 2.52±0.08
CBC9 6.89±0.31 4.63±0.32 2.49±0.25 7.23±0.64 4.89±0.32 2.48±0.05 7.98±0.32 5.85±0.25 2.36±0.09
CBC10 5.28±0.42 5.28±0.15 2.00±0.31 6.23±0.25 5.98±0.15 2.04±0.01 6.87±0.45 6.32±0.31 2.09±0.14
CBC11 10.26±0.36 4.34±0.13 3.36±0.21 13.5±0.45 4.89±0.45 3.76±0.04 15.26±0.42 5.78±0.56 3.64±0.21
CBC12 7.56±0.45 3.89±0.41 2.94±0.11 8.23±0.36 4.63±0.12 2.78±0.11 8.94±0.85 5.89±0.15 2.52±0.01
CBC13 14.26±0.12 4.26±0.25 4.35±0.14 16.25±0.15 4.79±0.75 4.39±0.12 18.25±0.45 5.64±0.16 4.24±0.04
CBC14 8.56±0.44 4.98±0.12 2.72±0.15 8.98±0.45 5.34±0.15 2.68±0.31 9.23±0.52 6.23±0.21 2.48±0.08
CBC15 15.29±0.12 5.1±0.44 4.00±0.31 18.26±0.12 5.84±0.16 4.13±0.15 19.25±0.21 6.28±0.85 4.07±0.04

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Table 6.16. Qualitative estimation of Potassium solubilization by selected isolates from Guava based
cropping system on Aleksandrov agar medium at 3 DAI, 5DAI, 7DAI

Potassium solubilization
Bacteria 3DAI 5DAI 7DAI
isolate
K halo Colony KSI K halo Colony KSI K halo Colony KSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
GBC1 5.26±0.21 5.24±0.21 2.00±0.11 5.89±0.42 5.98±0.21 1.98±0.11 6.23±0.24 6.25±0.45 2.00±0.11
GBC2 13.25±0.35 4.56±0.11 3.91±0.08 15.26±0.15 5.24±0.31 3.91±0.12 16.52±0.52 5.89±0.52 3.80±0.12
GBC3 5.26±0.14 5.28±0.11 2.00±0.05 6.25±0.16 5.89±0.45 2.06±0.13 7.25±0.26 6.25±0.63 2.16±0.13
GBC4 4.58±0.16 6.25±0.13 1.73±0.04 5.54±0.46 6.84±0.61 1.81±0.08 6.25±0.14 7.26±0.85 1.86±0.15
GBC5 5.64±0.15 4.85±0.12 2.16±0.01 6.25±0.14 5.26±0.48 2.19±0.08 7.26±0.35 6.48±0.45 2.12±0.14
GBC6 4.26±0.85 5.69±0.21 1.75±0.15 5.45±0.74 6.25±0.46 1.87±0.12 6.85±0.85 7.26±0.24 1.94±0.13
GBC7 7.56±0.15 4.89±0.24 2.55±0.12 8.25±0.85 5.48±0.12 2.51±0.11 9.25±0.25 5.89±0.63 2.57±0.11
GBC8 10.56±0.14 5.21±0.31 3.03±0.16 13.25±0.64 5.64±0.14 3.35±0.14 16.25±0.14 6.25±0.41 3.60±0.08
GBC9 2.56±0.63 3.54±0.25 1.72±0.13 3.48±0.63 5.26±0.08 1.66±0.12 4.58±0.31 6.45±0.85 1.71±0.09
GBC10 14.26±0.36 4.68±0.15 4.05±0.15 16.25±0.52 5.26±0.12 4.09±0.13 18.26±0.16 5.98±0.16 4.05±0.07
GBC11 5.96±0.85 5.26±0.81 2.13±0.14 6.23±0.25 6.25±0.31 2.00±0.17 7.26±0.42 6.89±0.31 2.05±0.12
GBC12 12.56±0.74 4.87±0.19 3.58±0.13 14.56±0.36 5.26±0.15 3.77±0.12 18.26±0.31 6.24±0.41 3.93±0.11
GBC13 5.28±0.16 4.63±0.16 2.14±0.15 6.23±0.48 4.98±0.14 2.25±0.15 7.26±0.52 6.14±0.25 2.18±0.07
GBC14 7.28±0.15 6.84±0.10 2.06±0.12 8.56±0.49 7.25±0.63 2.18±0.16 9.56±0.85 8.25±0.12 2.16±0.05
GBC15 9.56±0.16 5.46±0.16 2.75±0.21 10.25±0.32 6.25±0.12 2.64±0.21 13.25±.47 7.25±0.31 2.83±0.06
GBC16 5.84±0.25 4.85±0.12 2.20±0.13 6.25±0.11 5.85±0.15 2.07±0.11 7.26±0.16 6.32±0.74 2.15±0.11
GBC17 14.85±0.61 5.12±0.13 3.90±0.51 16.25±0.12 5.89±0.24 3.76±0.31 19.25±0.32 6.45±0.25 3.98±0.12
GBC18 4.85±0.84 4.63±0.14 2.05±0.12 5.26±0.31 5.47±0.32 1.96±0.14 7.26±0.48 6.25±0.12 2.16±0.14

Table 6.17. Qualitative estimation of Potassium solubilization by selected isolates from Dragon fruits-
based cropping system on Aleksandrov agar medium at 3 DAI, 5DAI, 7DAI
Potassium solubilization
Bacteria 3DAI 5DAI 7DAI
isolate K halo Colony KSI K halo Colony KSI K halo Colony KSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
DBC1 6.25±0.41 4.26±0.24 2.47±0.11 7.25±0.12 5.25±0.12 2.38±0.11 8.21±0.21 6.25±0.42 2.31±0.32
DBC2 8.56±0.32 3.58±0.36 3.39±0.15 9.25±0.14 4.25±0.13 3.18±0.02 10.24±0.31 5.48±0.24 2.87±0.85
DBC3 13.54±0.15 5.21±0.85 3.60±0.12 15.36±0.15 5.64±0.15 3.72±0.01 16.35±0.15 5.89±0.34 3.78±0.41
DBC4 16.45±0.16 4.89±0.51 4.36±0.13 17.26±0.13 5.48±0.14 4.15±0.15 18.25±0.14 5.98±0.15 4.05±0.34
DBC5 8.25±0.12 4.68±0.16 2.76±0.14 9.25±0.15 5.26±0.16 2.76±0.11 10.31±0.74 6.25±0.16 2.65±0.42
DBC6 3.45±0.15 5.26±0.75 1.66±0.11 4.25±0.15 6.24±0.12 1.68±0.13 5.26±0.85 6.89±0.14 1.76±0.34
DBC7 4.68±0.45 4.87±0.48 1.96±0.14 5.63±0.85 5.24±0.15 2.07±0.04 6.35±0.65 5.89±0.13 2.08±0.34
DBC8 10.85±0.85 4.26±0.46 3.55±0.11 12.65±0.15 4.89±0.14 3.59±0.05 16.35±0.14 5.26±0.14 4.11±0.45
DBC9 12.56±0.75 4.98±0.15 3.52±0.08 16.85±0.16 5.36±0.13 4.14±0.11 18.25±0.52 5.84±0.74 4.13±0.35
DBC10 8.56±0.63 5.26±0.12 2.63±0.09 10.25±0.85 5.84±0.14 2.76±0.13 10.98±0.31 6.11±0.16 2.80±0.85
DBC11 7.25±0.15 5.41±0.25 2.34±0.01 8.25±0.74 6.25±0.15 2.32±0.14 9.25±0.45 6.85±0.35 2.35±0.31
DBC12 6.58±0.45 4.82±0.63 2.37±0.12 7.56±0.65 6.25±0.11 2.21±0.16 8.25±0.36 7.2±0.25 2.15±0.34
DBC13 8.56±0.64 4.68±0.25 2.83±0.13 9.25±0.45 6.78±0.08 2.36±0.01 10.24±0.15 7.21±0.16 2.42±0.16
DBC14 11.56±0.15 5.1±0.85 3.27±0.14 13.54±0.26 5.26±0.09 3.57±0.06 16.25±0.15 5.89±0.14 3.76±0.25
DBC15 4.58±0.75 3.84±0.15 2.19±0.15 5.26±0.12 4.26±0.11 2.23±0.04 6.32±0.16 5.21±0.74 2.21±0.48
DBC16 6.54±0.15 4.26±0.23 2.54±0.13 7.25±0.85 5.36±0.12 2.35±0.01 8.24±0.85 6.21±0.16 2.33±0.12

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6.3.9. Bio-dissolution of insoluble zinc ores and release of Zinc

Zinc-solubilizing bacteria play a crucial role in converting zinc that is not easily
accessible in insoluble ores into a form that plants can effectively use in
agroecosystems. Their enzymes or acids degrade zinc molecules, rectifying soil
inadequacies and augmenting zinc absorption (Kushwaha et al. 2020). Zinc is
essential for the well-being of plants, since it plays a vital role in enzyme activity and
promotes development. These bacteria enhance agricultural productivity and
resistance, particularly in soils with zinc deficiency. This environmentally conscious
procedure diminishes dependence on artificial fertilizers, so fostering sustainable
agriculture (Shams Tabrez et al., 2021). Zinc-solubilizing bacteria play a crucial role
in releasing zinc from previously inaccessible sources, promoting the growth of strong
and healthy plants, and creating sustainable agricultural ecosystems. This
environmentally conscious method decreases dependence on chemical fertilizers and
advocates for sustainable agricultural techniques.

6.3.9.1. Plate assay

The study looked at the zinc solubilization capability of PGPR isolates in various
cropping systems and measured their effectiveness using Zinc Solubilizing Activity
Index (ZSI) values (Peng et al. 2020; Suriyachadkun, et al. 2022). In the banana
cropping system, BBC12 had the maximum zinc solubilizing activity, with ZSI values
ranging from 4.13 to 4.80 at different time intervals. For citrus, CBC8 showed the
greatest activity, with ZSI values ranging from 4.76 to 6.86. In the guava system,
GBC9 showed significant activity, with ZSI values ranging from 4.82 to 6.43. Finally,
in the dragon fruit system, DBC10 demonstrated significant action, with ZSI values
ranging from 4.12 to 5.81. These findings, presented in Tables 6.18 to 6.21,
demonstrate PGPR's broad zinc solubilization capabilities in a variety of agricultural
situations.

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Table 6.18. Qualitative estimation of Zink solubilization by selected isolates from Banana based
cropping system on Aleksandrov agar medium at 3 DAI, 5DAI, 7DAI

Zink solubilization
Bacteria 3DAI 5DAI 7DAI
isolate Zn halo Colony ZSI Zn halo Colony ZSI Zn halo Colony ZSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
BBC1 10.2±0.25 3.26±0.32 4.13±0.11 10.5±0.45 3.89±0.14 3.70±0.12 10.8±0.25 4.21±0.13 3.57±0.08
BBC2 8.26±0.36 4.26±0.45 2.94±0.21 9.25±0.16 5.21±0.12 2.78±0.11 10.35±0.65 5.34±0.25 2.94±0.09
BBC3 16.25±0.45 4.28±0.48 4.80±0.13 20.5±0.15 5.12±0.32 5.00±0.13 22.35±0.34 5.31±0.42 5.21±0.11
BBC4 10.26±0.63 3.32±0.36 4.09±0.15 12.3±0.78 3.78±0.15 4.25±0.31 13.2±048 4.21±0.32 4.14±0.12
BBC5 6.25±0.14 4.25±0.52 2.47±0.14 7.25±0.48 5.21±0.14 2.39±0.11 8.61±0.74 5.62±0.52 2.53±0.05
BBC6 5.26±0.25 4.85±0.45 2.08±0.31 6.25±0.65 5.32±0.85 2.17±0.12 7.21±0.46 5.42±0.63 2.33±0.06
BBC7 9.25±0.15 5.25±0.48 2.76±0.12 10.25±0.52 6.21±0.32 2.65±0.21 12.34±0.15 6.56±0.15 2.88±0.10
BBC8 13.52±0.75 4.23±0.11 4.20±0.14 16.35±0.41 4.39±0.15 4.72±0.11 19.23±0.45 4.89±0.14 4.93±0.05
BBC9 7.89±0.85 5.29±0.12 2.49±0.47 8.21±0.32 6.21±0.41 2.32±10 8.94±0.36 6.84±0.52 2.31±0.06
BBC10 10.25±0.45 3.25±0.15 4.15±0.25 12.34±0.15 4.25±0.52 3.90±0.12 13.2±0.15 5.24±0.35 3.52±0.11
BBC11 12.25±0.63 4.36±0.40 3.81±0.13 16.25±0.63 5.1±0.32 4.19±0.22 18.26±0.85 5.16±0.74 4.54±0.12
BBC12 18.26±0.15 4.89±0.16 4.73±0.15 24.25±0.41 5.13±0.14 5.73±0.24 24.85±0.45 5.42±0.85 5.58±0.02
BBC13 16.25±0.41 5.26±0.41 4.09±0.12 20.45±0.85 5.61±0.74 4.65±0.31 21.5±0.31 5.67±0.96 4.79±0.12
BBC14 8.56±0.52 4.63±0.25 2.85±0.15 9.26±0.32 5.32±0.15 2.74±0.25 10.3±0.34 5.34±0.63 2.93±0.21
BBC15 6.25±0.25 4.63±0.74 2.35±0.31 7.25±0.12 5.21±0.35 2.39±0.14 8.24±0.15 5.42±0.52 2.52±0.31
BBC16 7.29±0.63 3.25±0.25 3.24±0.15 8.26±0.15 4.23±0.15 2.95±0.15 8.65±0.32 4.65±0.42 2.86±0.21
BBC17 10.24±0.45 4.62±0.63 3.22±0.16 11.2±0.14 4.85±0.14 3.31±0.16 12.3±0.51 4.88±0.36 3.52±0.31
BBC18 13.25±0.52 5.21±0.89 3.54±0.32 15.3±0.32 5.38±0.36 3.84±0.11 16.2±0.16 5.27±0.41 4.07±0.14
BBC19 5.26±0.45 4.63±0.21 2.14±0.15 6.32±0.15 5.23±0.85 2.21±0.21 7.25±0.14 5.64±0.25 2.29±0.11
BBC20 8.26±0.48 4.56±0.12 2.81±0.14 9.25±0.11 4.89±0.63 2.89±0.14 10.2±0.85 5.84±0.31 2.75±0.10

Table 6.19. Qualitative estimation of Zink solubilization by selected isolates from Citrus based
cropping system on Aleksandrov agar medium at 3 DAI, 5DAI, 7DAI

Zink solubilization
Bacteria 3DAI 5DAI 7DAI
isolate Zn halo Colony ZSI Zn halo Colony ZSI Zn halo Colony ZSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
CBC1 15.23±0.25 5.26±0.63 3.90±0.11 17.23±0.35 5.36±0.21 4.21±0.11 17.89±0.31 5.48±0.12 4.26±0.11
CBC2 18.23±0.36 4.85±0.41 4.76±0.12 20.15±0.36 5.21±0.14 4.87±0.12 21.23±0.15 5.62±0.11 4.78±0.12
CBC3 20.14±0.45 4.58±0.25 5.40±0.11 24.26±0.85 4.89±0.13 5.96±0.13 26.25±0.34 4.98±0.16 6.27±0.13
CBC4 14.25±0.21 5.26±0.12 3.71±0.08 15.23±0.45 5.38±0.14 3.83±0.14 17.2±0.62 5.89±0.13 3.92±0.14
CBC5 11.23±0.35 4.36±0.52 3.58±0.06 13.25±0.63 4.89±0.16 3.71±0.15 13.25±0.14 5.21±0.14 3.54±0.08
CBC6 5.56±0.15 3.25±0.32 2.71±0.04 6.35±0.48 3.89±0.18 2.63±0.11 6.89±0.52 4.21±0.13 2.64±0.07
CBC7 8.56±0.41 4.26±0.14 3.01±0.01 10.25±0.45 4.98±0.11 3.06±0.32 11.2±0.15 5.12±0.14 3.19±0.09
CBC8 22.35±0.63 4.89±0.15 5.57±0.05 30.25±0.16 5.26±0.13 6.75±0.14 31.2±0.74 5.32±0.13 6.86±0.04
CBC9 24.26±0.85 5.21±0.25 5.66±0.11 28.25±0.12 5.64±0.15 6.01±0.17 29.2±0.65 5.72±0.14 6.10±0.05
CBC10 10.56±0.24 4.36±0.12 3.42±0.12 2.32±0.25 4.89±0.32 3.52±0.08 13.2±0.41 5.1±0.17 3.59±0.04
CBC11 13.56±0.63 3.85±0.13 4.52±0.12 14.25±0.14 4.67±0.15 4.05±0.09 15.32±0.25 4.72±0.35 4.25±0.07
CBC12 12.56±0.15 4.68±0.14 3.68±0.17 16.32±0.25 5.26±0.15 4.10±0.11 17.26±0.15 5.61±0.12 4.08±0.11
CBC13 9.85±0.32 5.36±0.41 2.84±0.11 10.45±0.15 5.84±0.32 2.79±0.02 12.3±0.14 6.2±0.31 2.98±0.12
CBC14 11.25±0.25 3.89±0.12 3.89±0.13 12.34±0.32 4.26±0.14 3.90±0.15 12.89±0.32 5.46±0.15 3.36±0.16
CBC15 16.25±0.63 4.25±0.13 4.82±0.15 19.35±0.85 4.87±0.11 4.97±0.13 21.3±0.15 5.74±.08 4.71±0.15

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Table 6.20. Qualitative estimation of Zink solubilization by selected isolates from Guava based
cropping system onAleksandrov agar medium at 3 DAI, 5DAI, 7DAI
Zink solubilization
Bacteria 3DAI 5DAI 7DAI
isolate Zn halo Colony ZSI Zn halo Colony ZSI Zn halo Colony ZSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
GBC1 8.6±0.85 3.25±0.21 3.65±0.11 10.2±0.21 4.32±0.32 3.36±0.11 11.23±0.14 4.51±0.11 3.49±0.32
GBC2 12.25±0.41 4.25±0.15 3.88±0.09 13.25±0.14 4.89±0.14 3.71±0.18 15.26±0.25 5.32±0.12 3.87±0.52
GBC3 15.3±0.63 3.85±0.13 4.98±0.05 21.0±0.42 4.26±0.12 5.93±0.09 25.23±0.26 4.89±0.25 6.16±0.08
GBC4 7.26±0.41 4.21±0.14 2.72±0.05 8.21±0.52 5.21±0.13 2.58±0.08 9.25±0.23 5.89±0.31 2.57±0.07
GBC5 9.25±0.75 4.36±0.16 3.12±0.07 10.2±0.63 5.31±0.32 2.92±0.11 11.23±0.85 5.64±0.14 2.99±0.11
GBC6 11.2±0.65 3.85±0.13 3.91±0.07 12.34±0.74 4.26±0.15 3.90±0.12 14.22±0.45 5.89±0.32 3.41±0.12
GBC7 10.36±0.42 5.34±0.15 2.94±0.11 13.25±0.16 5.64±0.32 3.35±0.13 14.25±0.41 5.78±0.15 3.47±0.32
GBC8 16.25±0.12 3.89±0.17 5.18±0.02 23.35±0.85 4.32±0.14 6.41±0.18 25.63±0.52 4.89±0.45 6.24±0.11
GBC9 18.24±0.63 4.21±0.85 5.33±0.31 25.36±0.41 4.89±0.45 6.19±0.08 28.24±0.32 5.21±0.15 6.42±0.15
GBC10 20.36±0.41 3.75±0.96 6.43±0.01 23.52±0.32 4.35±0.63 6.41±0.07 26.34±0.15 5.12±0.32 6.14±0.11
GBC11 19.54±0.85 5.12±0.15 4.82±0.21 26.24±0.14 5.42±0.17 5.84±0.14 27.89±0.63 5.64±0.15 5.95±0.12
GBC12 6.35±0.32 4.23±0.16 2.50±0.14 8.26±0.52 5.32±0.85 2.55±0.15 9.24±0.41 5.35±0.11 2.73±0.01
GBC13 5.24±0.15 3.85±0.15 2.36±0.13 6.45±0.13 3.89±0.25 2.66±0.14 8.2±0.85 4.26±0.12 2.92±0.08
GBC14 8.6±0.42 4.26±0.32 3.02±0.19 10.26±0.12 5.24±0.12 2.96±0.35 12.23±0.15 5.64±0.13 3.17±0.06
GBC15 10.56±0.32 3.75±0.15 3.82±0.14 12.35±0.14 4.23±0.32 3.92±0.15 13.52±032 5.26±0.14 3.57±0.12
GBC16 11.34±0.15 4.16±0.12 3.73±0.32 13.25±0.85 4.26±0.15 4.11±0.11 14.62±0.14 5.34±0.14 3.74±0.05
GBC17 9.84±0.85 3.48±0.21 3.83±0.15 10.52±0.41 4.85±0.12 3.17±0.11 11.25±0.35 5.63±0.04 3.00±0.56
GBC18 10.5±0.12 4.23±0.14 3.48±0.32 11.25±0.12 5.21±0.11 3.16±0.07 13.25±0.41 6.2±0.08 3.14±0.45

Table 6.21. Qualitative estimation of Zink solubilization by selected isolates from Dragon fruits-based
cropping system onAleksandrov agar medium at 3 DAI, 5DAI, 7DAI

Zink solubilization
Bacteria 3DAI 5DAI 7DAI
isolate Zn halo Colony ZSI Zn halo Colony ZSI Zn halo Colony ZSI
zone (mm) diameter zone (mm) diameter zone (mm) diameter
(mm) (mm) (mm)
DBC1 10.25±0.24 5.21±0.21 2.97±0.11 11.25±0.24 5.31±0.32 3.12±0.11 12.31±0.42 5.35±0.14 3.30±0.11
DBC2 8.26±0.45 4.28±0.63 2.93±0.12 9.26±0.26 4.89±0.32 2.89±0.05 10.25±0.63 4.98±0.31 3.06±0.15
DBC3 5.26±0.26 4.36±0.75 2.21±0.13 6.25±0.23 4.67±0.42 2.34±0.08 6.89±0.15 4.87±0.12 2.41±0.08
DBC4 6.85±0.25 5.12±0.56 2.34±0.14 7.25±0.52 5.31±0.23 2.37±0.11 8.25±0.15 5.89±0.15 2.40±0.09
DBC5 10.34±0.32 3.25±0.45 4.18±0.11 11.24±0.41 4.32±0.15 3.60±0.11 13.25±0.16 5.21±0.13 3.54±0.11
DBC6 13.25±0.85 4.25±0.32 4.12±0.08 16.25±0.25 4.58±0.16 4.55±0.5 20.14±0.14 5.14±0.14 4.92±0.12
DBC7 8.65±0.14 5.15±0.52 2.68±0.11 10.25±0.85 5.65±0.14 2.81±0.14 12.35±0.32 6.2±0.13 2.99±0.13
DBC8 9.45±0.25 4.85±0.41 2.95±0.09 12.24±0.63 4.89±0.36 3.50±0.01 14.25±0.25 5.12±0.15 3.78±0.05
DBC9 7.26±0.14 5.23±0.63 2.39±0.05 8.56±0.14 5.7±0.58 2.50±0.05 9.56±0.16 6.23±0.14 2.53±0.08
DBC10 15.26±0.36 4.85±0.74 4.15±0.12 19.25±0.15 5.21±0.15 4.69±0.05 23.25±0.19 5.42±0.21 5.29±0.11
DBC11 17.25±0.36 4.96±0.25 4.48±0.20 21.25±0.14 5.31±0.16 5.00±0.11 25.21±0.12 5.61±0.16 5.49±0.14
DBC12 12.52±0.74 4.57±0.32 3.74±0.15 15.35±0.74 5.12±0.32 4.00±0.12 19.25±0.13 5.32±0.18 4.62±0.32
DBC13 16.25±0.85 3.89±0.14 5.18±0.14 20.36±0.85 4.23±0.14 5.81±0.12 24.23±0.15 5.12±0.13 5.73±0.12
DBC14 10.5±0.96 5.21±0.16 3.02±0.12 12.32±0.36 5.64±0.15 3.18±0.04 14.25±0.18 5.84±0.12 3.44±0.11
DBC15 3.25±0.41 4.26±0.63 1.76±0.13 4.25±0.84 5.24±0.85 1.81±0.12 5.56±0.32 5.34±0.15 2.04±0.12
DBC16 4.25±0.52 5.12±0.14 1.83±0.10 6.5±0.63 5.86±0.15 2.11±0.13 8.2±0.25 6.23±0.16 2.32±0.13

6.3.9.1. Broth assay of phosphate solubilization of PGPR isolate from Banana

based cropping system
Using broth assay, phosphate-solubilizing bacteria from various crops are tested.
These bacteria are inoculated into liquid medium with insoluble phosphates, which
convert into soluble forms. This method quantifies phosphorus release, essential for
plant uptake (Aliyat et al. 2022). Understanding the effectiveness of these bacteria can

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help select beneficial strains, improve soil fertility, and reduce agricultural chemical
fertilizer use. This study subjects five bacteria to varied levels of tricalcium phosphate
(TCP) for five days to determine their phosphate solubilization ability in a diverse
agricultural environment. The bacteria are cultivated with different levels of
tricalcium phosphate (TCP) to test their ability to convert insoluble phosphate into
plant-usable forms. Plants in the diversified cropping system affect bacterial activity.
Assessing soluble phosphate production reveals bacteria efficiency. The research
seeks bacterium strains that dissolve phosphate well in diverse agricultural systems,
even when phosphate levels alter. This helps improve agriculture and plant nutrition
through microbial interactions.

6.3.9.2. Broth assay of phosphate solubilization of PGPR isolate from Banana

based cropping system
The efficacy of phosphate solubilization varied depending on the concentration of
tricalcium phosphate (TCP) and the period of incubation. At TCP concentrations of
2.5 g/L, phosphate solubilization efficiency ranged from 110 to 385 μg/mL across
incubation periods of 24 to 120 hours. Increasing TCP concentration to 5 g/L resulted
in a range of 220 to 695 μg/mL, while at 7.5 g/L, the range was 320 to 695 μg/mL,
and at 10 g/L, it was 355 to 725 μg/mL. These values show bacterial isolates' ability
to solubilize phosphate over time, with higher TCP concentrations often resulting in
greater solubilization efficiency. The findings, presented in Table 6.22, shed light on
the temporal dynamics of phosphate solubilization and its impact on pH changes
(Yadav et al. 2023).

6.3.9.3. Broth assay of phosphate solubilization of PGPR isolate from Citrus

based cropping system
The study, conducted in a citrus-based cropping system, investigated the phosphate
solubilization effectiveness of bacterial strains CBC4, CBC5, CBC7, CBC10, and
CBC15 at various tricalcium phosphate (TCP) concentrations and incubation periods.
The results showed substantial differences in phosphate solubilization efficiency at
TCP doses of 2.5 g/L, 5 g/L, 7.5 g/L, and 10 g/L across incubation times ranging from
24 to 120 hours. The results, reported in Table 6.23, illustrate the phosphate solubility
efficiency at a TCP concentration of 2.5 g/L, the phosphate solubilization efficiency
varied from 150 to 350 μg/mL after 24 hours of incubation and increased to 310-520
μg/mL after 120 hours. Similarly, at TCP concentrations of 5 g/L, 7.5 g/L, and 10 g/L,

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the phosphate solubilization efficiency shown rising trends over time, with values
ranging from 210 to 410 μg/mL, 310 to 510 μg/mL, and 420 to 640 μg/mL,
respectively, after 24 hours, and reaching higher ranges after 120 hours of incubation.

6.3.9.4. Broth assay of phosphate solubilization of PGPR isolate from Guava

based cropping system
In a Guava-based cultivation system, bacterial strains GBC1, GBC4, GBC7, GBC8,
and GBC11 demonstrated significant phosphate solubilization efficiency across
various tricalcium phosphate (TCP) concentrations and time intervals. The results,
reported in Table 6.24. the phosphate solubilization efficiency ranged from 130 to 740
μg/mL, indicating a substantial capacity to release phosphate into the medium.
Notably, at a TCP concentration of 2.5 g/L, GBC8 exhibited the highest phosphate
solubilization efficiency, reaching up to 420 μg/mL after 120 hours of incubation.
These critical values highlight the potential of these bacterial strains to profoundly
impact phosphate availability in Guava cultivation, emphasizing their importance in
agricultural practices.

6.3.9.5. Broth assay of phosphate solubilization of PGPR isolate from Dragon

fruits-based cropping system
The investigation in Dragon-fruit agriculture revealed varying phosphate
solubilization efficiencies among bacterial strains DBC5, DBC6, DBC7, DBC10, and
DBC12 across different TCP concentrations and incubation durations. At 2.5 g/L TCP,
DBC5 demonstrated the highest solubilization efficiency, followed by DBC12 and
DBC7. The result reported in Table Table 6.25. that show the Efficiency increased
with time, ranging from 110 to 215 (μg/mL) after 24 hours to 270 to 410 (μg/mL)
after 120 hours. Similar trends were observed at higher TCP concentrations, with
DBC5 consistently exhibiting superior efficiency. The experiment also noted pH
variations, indicative of bacterial activity-induced organic acid generation. These
findings suggest DBC5's potential for enhancing phosphate solubilization in Dragon-
fruit agriculture, with efficiency influenced by TCP concentration and incubation
duration (Goswami et al. 2019).

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Table 6.22. Phosphate solubilization efficiency of Five promising bacteria of banana-based cropping system with different TCP concentrations (Incubation for 5 days).
*Values are Mean ±SD of triplicate values, *TCP - Tricalcium Phosphate

Bacterial Conc. of *PO4 2- Solubilized (in μg/mL) *Change of pH

isolate TCP g/L (Initial pH of media 6.8)
24hrs 48hrs 72hrs 96hrs 120hrs 24hrs 48hrs 72hrs 96hrs 120hrs
BBC4 2.5 125±12.5 210±14.2 230±10.2 245±15.23 385±18.56 4.26±0.24 4.28±0.26 4.29±0.08 4.31±0.12 4.34±0.13
5.0 220±10.25 240±21.23 270±18.56 310±23.25 320±22.85 4.27±0.12 4.29±0.16 4.31±0.13 4.34±0.15 4.36±0.14
7.5 425±25.2 450±28.56 520±30.2 540±26.56 510±32.52 4.30±0.08 4.32±0.11 4.35±0.16 4.31±0.18 4.38±0.16
10.0 510±27.25 530±34.25 580±36.56 550±34.85 542±40.26 4.34±0.15 4.35±0.21 4.37±0.15 4.38±0.16 4.39±0.19
BBC9 2.5 110±12.23 255±13.25 245±16.53 320±18.26 340±21.23 4.46±0.15 4.47±0.06 4.48±0.14 4.50±0.16 4.52±0.14
5.0 230±14.25 240±15.23 280±20.15 350±21.25 410±23.36 4.43±0.08 4.45±0.07 4.46±0.15 4.48±0.12 4.46±0.13
7.5 320±18.36 350±15.26 380±20.36 420±17.26 540±21.26 4.44±0.12 4.45±0.11 4.46±0.16 4.48±0.14 4.47±0.12
10.0 420±11.25 450±15.26 476±14.26 520±18.25 600±16.25 4.45±0.16 4.46±0.18 4.43±0.13 4.46±0.14 4.46±0.13
BBC11 2.5 180±30.25 190±32.25 245±34.26 295±38.26 310 ±34.25 4.12±0.11 4.14±0.15 4.15±0.16 4.16±0.15 4.21±0.14
5.0 315±10.23 342±18.56 380±19.26 410±15.26 435±14.26 4.15±0.12 4.16±0.14 4.17±0.17 4.20±0.12 4.24±0.15
7.5 350±25.26 370±22.23 390±21.23 405±26.36 410±15.26 4.17±0.21 4.18±0.24 4.16±0.23 4.17±0.21 4.15±0.14
10.0 355± 18.5 380±16.23 392±17.26 415±19.56 428 ±21.25 4.13±0.16 4.12±0.23 4.13±0.25 4.10±0.16 4.09±0.12
BBC15 2.5 150±10.56 158±11.25 170±13.25 210±14.26 220±18.23 5.12±0.21 5.16±0.15 5.18±0.18 5.21±0.12 5.22±0.14
5.0 280±12.23 295±14.26 310±16.52 320±17.26 340±15.26 5.14±0.10 5.16±0.14 5.17±0.15 5.20±0.16 5.23±0.25
7.5 430±13.35 450±12.26 470±15.23 472±14.26 510±18.26 5.16±0.23 5.17±021 5.21±0.23 5.22±0.24 5.24±0.12
10.0 410±19.23 430±20.12 475±17.26 490±12.23 520±14.25 5.21±0.12 5.23±0.31 5.26±0.14 5.27±0.16 5.18±0.16
BBC18 2.5 210±25.23 240±24.23 263±28.56 280±24.26 320±28.26 6.61±0.12 6.62±0.25 6.64±0.26 6.63±0.16 6.65±0.15
5.0 265±21.23 280±23.23 310±20.25 340±26.23 360±27.26 6.63±0.23 6.64±0.21 6.61±0.23 6.62±0.24 6.60±0.12
7.5 510±18.26 550±19.56 560±24.26 610±16.23 695±17.25 6.60±0.15 6.58±0.15 6.57±0.16 6.64±0.16 6.64±0.21
10.0 645±25.23 660±24.26 680±29.36 710±24.26 725±27.26 6.45±0.16 6.42±0.25 6.41±0.21 6.43±0.10 6.40±0.13

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Table 6.23. Phosphate solubilization efficiency of Five promising bacteria of Citrus-based cropping system with different TCP concentrations (Incubation for 5 days) *Values
are Mean ±SD of triplicate values, *TCP - Tricalcium Phosphate

Bacterial isolate Conc. of TCP g/L *PO4 2- Solubilized (in μg/mL) *Change of pH
(Initial pH of media 6.8)
24hrs 48hrs 72hrs 96hrs 120hrs 24hrs 48hrs 72hrs 96hrs 120hrs

CBC4 2.5 150±10.12 220±11.35 260±16.35 260±14.45 415±15.85 5.23±0.12 5.25±0.13 5.27±0.18 5.30±0.21 5.31±0.14
5.0 250±11.52 350±16.25 420±10.25 450±8.96 510±11.25 5.25±0.15 5.27±0.15 5.28±0.16 5.32±0.23 5.34±0.16
7.5 310±17.15 360±12.25 410±11.25 450±10.2 620±9.56 5.26±0.14 5.28±0.18 5.31±0.16 5.25±0.25 5.34±0.15
10.0 450±13.25 468±10.26 510±13.35 560±10.28 640±11.28 5.31±0.23 5.32±0.17 5.34±0.14 5.36±0.14 5.32±0.18
CBC5 2.5 230±7.08 240±17.25 280±10.56 310±9.58 410±6.58 4.62±0.15 4.60±0.15 4.61±0.12 4.63±0.23 4.65±0.35
5.0 310±10.24 350±12.25 410±16.32 480±14.26 520±10.25 4.63±0.11 4.62±0.12 4.65±0.14 4.64±0.21 4.67±0.15
7.5 450±11.25 470±13.56 520±18.56 590±14.26 630±12.23 4.65±0.16 4.67±0.13 4.68±0.16 4.67±0.31 4.62±0.12
10.0 520±17.26 540±12.26 620±10.25 650±9.25 710±15.23 4.63±0.14 4.62±0.13 4.60±0.15 4.58±0.45 4.57±0.36
CBC7 2.5 350±20.13 370±25.23 410±21.23 480±15.26 520±16.23 6.31±0.13 6.32±0.25 6.34±0.20 6.33±0.56 6.30±0.45
5.0 410±17.26 450±15.26 480±18.26 560±20.23 570±19.26 6.36±0.12 6.34±0.14 6.33±0.21 6.31±0.12 6.30±0.42
7.5 510±15.23 540±14.26 570±12.23 642±14.25 710±16.23 6.33±0.10 6.32±0.15 6.31±0.56 6.30±0.14 6.28±0.14
10.0 640±23.12 670±21.31 750±12.03 780±15.26 810±12.30 6.30±0.13 6.29±0.13 6.29±0.14 6.30±0.16 6.31±0.12
CBC10 2.5 180±6.89 210±10.25 240±7.89 270±10.26 310±11.23 4.31±0.07 4.32±0.15 4.34±0.63 4.33±0.12 4.30±0.31
5.0 210±16.23 245±14.25 286±12.23 345±10.25 380±16.23 4.32±0.62 4.36±0.17 4.32±0.32 4.31±0.31 4.32±0.36
7.5 345±15.23 372±14.26 410±12.23 490±18.26 520±21.25 4.30±0.12 4.31±0.16 4.29±0.12 4.28±0.12 4.27±0.14
10.0 440±17.25 480±14.25 580±12.23 650±11.23 710±10.26 4.36±0.16 4.35±0.11 4.33±0.14 4.36±0.32 4.32±0.12
CBC15 2.5 240±10.25 260±13.26 345±12.25 362±16.52 380±18.25 4.12±0.12 4.15±0.18 4.17±0.07 4.18±0.21 4.21±0.35
5.0 280±16.23 310±14.25 345±18.26 380±17.26 410±16.23 4.14±0.26 4.16±0.23 4.17±0.32 4.17±0.31 4.19±0.14
7.5 350±10.25 380±10.25 420±11.26 510±14.25 580±13.25 4.16±0.35 4.18±0.86 4.21±0.63 4.22±0.12 4.23±0.36
10.0 420±11.25 485±18.25 580±16.35 620±14.36 680±13.26 4.20±0.48 4.23±0.15 4.24±0.31 4.21±0.15 4.19±0.14

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Table 6.24. Phosphate solubilization efficiency of Five promising bacteria of Guava-based cropping system with different TCP concentrations (Incubation for 5 days)
*Values are Mean ±SD of triplicate values, *TCP - Tricalcium Phosphate

Bacterial Conc. *PO4 2- Solubilized (in μg/mL) *Change of pH

isolate of TCP (Initial pH of media 6.8)
g/L 24hrs 48hrs 72hrs 96hrs 120hrs 24hrs 48hrs 72hrs 96hrs 120hrs
GBC1 2.5 130± 20.15 160± 12.35 180±14.23 210±21.36 230±15.26 6.12±0.24 6.15±0.12 6.18±0.12 6.21±0.12 6.25±0.14
5.0 150± 12.35 262±16.23 290±12.32 310±20.36 350±10.25 6.15±0.12 6.23±0.11 6.22±0.16 6.24±0.15 6.29±0.15
7.5 310±15.56 340±14.26 410±12.25 480±12.23 510±14.26 6.17±0.12 6.19±0.08 6.21±0.14 6.25±0.14 6.23±0.26
10.0 520±21.25 580±12.35 610±13.35 620±10.36 630±13.35 6.18±0.13 6.21±0.06 6.23±0.05 6.27±0.16 6.29±0.17
GBC4 2.5 210±13.36 240±18.26 280±12.53 340±11.25 350±17.26 5.28±0.04 5.29±0.16 5.31±0.25 5.30±0.12 5.29±0.23
5.0 250±15.24 310±14.26 345±11.25 410±12.36 480±8.26 5.29±0.05 5.29±0.18 5.30±0.14 5.32±0.13 5.31±0.14
7.5 385±16.34 452±16.32 496±13.25 570±14.25 620±12.50 5.30±0.12 5.31±0.19 5.32±0.03 5.33±0.08 5.32±0.08
10.0 620±18.26 640±21.25 680±16.36 710±10.36 740±16.36 5.30±0.16 5.31±0.04 5.33±0.41 5.34±0.08 5.31±0.12
GBC7 2.5 185±21.35 250±16.32 270±12.35 310±13.35 350±14.25 4.23±0.15 4.24±0.15 4.25±0.96 4.26±0.14 4.25±0.12
5.0 270±20.36 345±18.26 380±17.25 410±11.25 480±13.35 4.24±0.12 4.26±0.15 4.25±0.45 4.27±0.16 4.26±0.4
7.5 450±24.25 480±21.25 510±13.36 570±13.36 610±10.25 4.26±0.16 4.26±0.16 4.26±0.15 4.29±0.17 4.28±0.36
10.0 520±19.25 580±14.26 640±15.26 680±15.26 710±11.23 4.27±0.21 4.30±0.17 4.29±0.14 4.31±0.15 4.32±0.45
GBC8 2.5 230±21.25 310±16.32 380±13.36 410±10.23 420±14.26 4.11±0.25 4.12±0.19 4.12±0.15 4.13±0.16 4.16±0.35
5.0 245±12.36 345±15.26 400±14.25 415±18.26 456±13.35 4.12±0.14 4.13±0.12 4.14±0.12 4.13±0.13 4.17±0.17
7.5 260±14.25 365±12.36 430±21.36 448±21.25 510±18.96 4.14±0.14 4.15±0.13 4.12±0.14 4.13±0.85 4.16±0.36
10.0 280±18.26 395±10.25 450±20.36 480±10.23 535±20.31 4.13±0.36 4.12±0.07 4.12±0.36 4.14±0.14 4.12±0.12
GBC11 2.5 132±23.25 185±14.25 210±21.36 275±14.23 289±21.23 6.43±0.14 6.42±0.06 6.47±0.17 6.45±0.23 6.44±0.15
5.0 189±24.26 245±16.23 249±14.25 290±13.23 350±15.26 6.42±0.17 6.41±0.15 6.40±0.26 6.41±0.36 6.42±0.17
7.5 220±21.25 245±12.23 278±16.23 340±10.25 389±14.23 6.40±0.16 6.39±0.02 6.38±0.15 6.41±0.14 6.42±012
10.0 250±14.25 290±10.36 320±10.20 350±11.25 450±10.23 6.41±0.16 6.42±0.04 6.43±0.12 6.41±0.17 6.38±0.15

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Table 6.25. Phosphate solubilization efficiency of Five promising bacteria of Dragon fruits -based cropping system with different TCP concentrations (Incubation for 5 days)
*Values are Mean ±SD of triplicate values, *TCP - Tricalcium Phosphate

Bacterial Conc. *PO4 2- Solubilized (in μg/mL) *Change of pH

isolate of TCP (Initial pH of media 6.8)
g/L 24hrs 48hrs 72hrs 96hrs 120hrs 24hrs 48hrs 72hrs 96hrs 120hrs
DBC5 2.5 215±20.12 250±14.25 290±15.23 320±10.32 350±15.23 5.12±0.26 5.16±0.45 5.32±0.36 5.35±0.85 5.42±0.09
5.0 280±15.12 310±12.23 350±12.23 380±13.36 420±14.23 5.13±0.05 5.18±0.14 5.24±0.15 5.40±0.46 5.46±0.08
7.5 310±14.23 350±15.25 410±10.25 460±14.25 523±10.23 5.16±0.08 5.26±0.16 5.28±0.36 5.31±0.15 5.35±0.05
10.0 350±15.23 380±14.23 405±14.23 480±17.23 540±17.29 5.16±0.15 5.12±0.18 5.32±0.32 5.34±0.75 5.40±0.15
DBC6 2.5 110±10.23 190±12.23 240±17.25 260±16.23 310±15.26 6.53±0.16 6.51±0.18 6.50±0.14 6.52±0.16 6.54±0.16
5.0 190±17.26 250±14.23 290±13.36 340±13.35 360±13.35 6.54±0.15 6.54±0.16 6.55±0.18 6.53±0.32 6.56±0.16
7.5 210±16.23 280±10.23 340±15.25 420±11.23 480±10.25 6.56±0.16 6.54±0.14 6.55±0.36 6.53±0.85 6.57±0.13
10.0 280±15.23 345±14.23 395±13.65 510±13.36 540±11.36 6.54±0.25 6.52±0.96 6.51±0.96 6.54±0.16 6.53±0.17
DBC7 2.5 180±10.45 210±17.25 230±24.23 250±10.25 270±16.23 4.25±0.26 4.23±0.36 4.21±0.25 4.23±0.15 4.21±0.12
5.0 210±18.63 250±18.23 280±21.25 340±12.15 380±12.35 4.23±0.36 4.24±0.14 4.21±0.14 4.26±0.32 4.25±0.08
7.5 240±13.25 270±10.23 320±19.25 380±13.36 420±10.25 4.25±0.14 4.23±0.18 4.25±0.25 4.24±0.16 4.24±0.85
10.0 290±21.23 350±15.23 410±21.36 460±14.23 490±14.25 4.26±0.85 4.25±0.16 4.23±0.32 4.21±0.16 4.22±0.64
DBC10 2.5 125±10.23 180±10.36 250±14.25 280±13.35 300±17.26 4.58±0.25 4.56±0.32 4.57±0.18 4.52±0.12 4.51±0.46
5.0 150±15.23 240±17.25 290±13.45 320±16.32 350±13.35 4.59±0.39 4.57±0.14 4.56±0.19 4.58±0.32 4.55±0.23
7.5 180±10.26 295±16.23 345±12.36 450±14.25 570±16.35 4.62±0.48 4.62±0.47 4.65±0.14 4.63±0.16 4.67±0.85
10.0 250±15.23 345±15.23 450±14.23 589±21.23 650±17.25 4.64±0.15 4.63±0.23 4.63±0.25 4.68±0.12 4.7±0.16
DBC12 2.5 210±13.23 280±10.25 316±18.25 385±20.36 410±10.23 4.98±0.09 5.1±0.18 4.96±0.16 4.99±0.16 5.2±0.75
5.0 240±16.23 318±11.25 390±14.25 450±25.36 520±11.62 5.1±0.05 5.12±0.15 5.13±0.38 5.16±0.18 5.18±0.16
7.5 320±18.26 387±10.36 456±13.36 570±20.36 610±10.25 5.14±0.15 5.12±0.14 5.16±0.23 5.18±0.08 5.20±0.85
10.0 340±14.26 415±18.26 475±12.23 610±13.25 670±18.25 5.15±0.18 5.16±0.16 5.14±0.18 5.21±0.10 5.26±0.34

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6.3.9.6. Broth assay of IAA production of PGPR isolate from Banana based
cropping system
The study looked at how different tryptophan concentrations (from 0.0 to 1 g/L)
affected Indole-3-acetic acid (IAA) synthesis in bacteria that are essential for plant
growth. Higher tryptophan concentrations significantly boosted IAA synthesis, with
an ideal level for maximum efficiency. Table 6.26. reported that Bacterial strains
BBC7, BBC15, and BBC5 showed considerable IAA production at 0.0 g/L
tryptophan, ranging from 16.2 to 28.9 (μg/mL) over 24–96 hours. With 0.3 g/L
tryptophan, IAA synthesis ranged from 17.5 to 30.1 (μg/mL) throughout the same
period. At 0.7 g/L and 1 g/L tryptophan, IAA production efficiency ranged from 18.2
to 30.6 (μg/mL) and 17.8 to 29.9 (μg/mL), respectively, over the incubation durations.
The study also revealed dynamic pH fluctuations over time caused by bacterial
metabolism, emphasizing the complex interplay between tryptophan concentration,
bacterial activity, and IAA production, crucial for optimizing agricultural practices
and enhancing crop yield.
6.3.9.7. Broth assay of IAA production of PGPR isolate from Citrus based
cropping system
The study looked at how tryptophan affected bacterial Indole-3-acetic acid (IAA)
synthesis during a 24-96-hour period. The Table 6.27. reported that at 0.0 g/L
tryptophan, IAA levels ranged from 19.6 to 26.5 (μg/mL), with CBC12 demonstrating
the highest synthesis, followed by CBC13 and CBC3. At 0.3 g/L, IAA levels varied
from 19.8 to 29.2 (μg/mL). 0.7 g/L ranged from 19.3 to 28.1 (μg/mL), whereas 1 g/L
ranged from 18.2 to 27.8 (μg/mL). Initial tryptophan breakdown caused minor pH
reductions, which were exacerbated by bacterial growth within 48 hours. By 72 hours,
considerable bacterial activity had created noticeable pH changes, which were
influenced by organic acid buildup and resource depletion.

6.3.9.8. Broth assay of IAA production of PGPR isolate from Guava based
cropping system
The study looked at IAA synthesis in bacteria at various tryptophan doses for 24 to 96
hours. The Table 6.28. find that at 0.0 g/L tryptophan, IAA levels ranged from 15.2 to
36.32 (μg/mL), with GBC12 demonstrating the highest synthesis, followed by GBC3
and GBC16. At 0.3 g/L tryptophan, IAA ranged from 16.2 to 35.25 (μg/mL), while at

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0.7 g/L it ranged from 18.2 to 33.4 (μg/mL). With 1 g/L tryptophan, IAA synthesis
ranged from 18.1 to 33.7 (μg/mL). Initial tryptophan breakdown resulted in minor pH
decreases, which were exacerbated within 48 hours by enhanced bacterial
proliferation, influencing subsequent pH values and stability.
6.3.9.9. Broth assay of IAA production of PGPR isolate from Dragon fruits-based
cropping system
Over the course of 24 to 96 hours, the researchers detected IAA synthesis in bacteria
at various tryptophan concentration levels. The Table 6.29. find that at 0.0 g/L
tryptophan, IAA levels ranged from 11.25 to 23.5 (μg/mL), with DBC14 showing the
highest synthesis, followed by DBC11 and DBC6. At 0.3 g/L tryptophan, IAA ranged
from 13.85 to 24.2 (μg/mL), while at 0.7 g/L it ranged from 14.36 to 25.6 (μg/mL).
With 1 g/L tryptophan, IAA synthesis ranged from 15.2 to 25.3 (μg/mL). Initial
tryptophan breakdown resulted in minor pH decreases, which were exacerbated
within 48 hours by enhanced bacterial proliferation, influencing subsequent pH values
and stability.

Table 6.26. Amount of IAA produced in μg/mL by Five promising bacteria of Banana-based cropping
system with different concentration of Tryptophan.
Conc. of *Amount of IAA Produced in μg /mL *Change of pH
Bacterial Tryptophan 24hrs 48hrs 72hrs 96hrs 24hrs 48hrs 72hrs 96hrs
isolate g/L
BBC2 0 16.2±0.85 18.2±0.15 16.3±2.1 12.2±0.45 6.7±0.25 6.4±0.74 5.2±0.74 4.8±0.14
0.3 19.2±0.15 19.3±0.25 19.6±0.85 19.9±0.26 6.6±0.85 5.8±0.41 5.4±0.31 4.9±0.25
0.7 21.2±0.86 21.3±0.78 21.5±0.56 22.1±0.74 6.7±0.45 6.2±0.63 5.8±0.32 4.4±0.34
1 18.4±0.14 19.2±0.89 20.1±0.42 20.4±0.63 6.7±0.14 6.3±0.18 5.4±0.14 4.6±0.14
BBC7 0 28.3±1.2 28.5±0.25 28.6±0.12 28.9±0.45 6.8±0.15 6.2±0.15 5.9±0.16 4.8±0.74
0.3 29.4±2.1 29.6±0.85 29.7±0.36 30.1±0.25 6.7±0.26 5.9±0.09 5.3±0.85 4.4±0.52
0.7 30.1±3.5 29.8±0.56 30.5±0.25 30.4±0.14 6.7±0.42 6.2±0.10 6.4±0.12 6.6±0.31
1 28.2±0.25 28.9±0.47 29.6±0.45 29.9±1.25 6.8±0.31 6.4±0.63 5.8±0.31 4.8±0.15
BBC10 0 17.2±0.15 17.6±0.16 17.9±1.25 18.1±0.85 6.9±0.74 6.6±0.01 6.2±0.12 6.1±0.16
0.3 17.5±0.56 17.8±1.2 18.2±1.65 18.4±1.36 6.8±0.21 6.7±0.5 6.1±0.12 5.8±0.14
0.7 18.2±0.75 18.6±4.6 18.9±2.13 19.2±1.85 6.6±0.15 6.1±0.06 5.8±0.70 5.1±0.12
1 17.8±0.56 17.9±6.5 19.2±0.25 19.5±1.45 6.7±0.36 6.5±0.13 6.6±0.13 6.2±0.62
BBC15 0 21.2±0.85 21.5±2.3 21.4±0.34 21.7±1.36 6.9±0.17 6.7±0.13 6.8±0.16 6.6±0.13
0.3 21.6±0.36 21.8±4.2 21.9±0.85 22.4±2.15 6.8±0.96 6.1±0.7 5.9±0.11 5.7±0.85
0.7 22.2±0.56 22.3±6.3 22.5±0.96 22.7±1.4 6.7±0.12 6.4±0.31 6.3±0.08 6.1±0.21
1 21.8±1.25 22.1±5.2 22.6±0.34 22.4±1.35 6.7±0.31 6.6±0.18 6.3±0.06 5.9±0.24
BBC18 0 18.2±2.2 18.4±7.5 18.6±0.45 18.7±2.14 6.7±0.25 6.1±0.25 5.8±0.12 5.7±0.16
0.3 18.4±4.5 18.6±1.2 18.8±0.75 19.1±1.23 6.8±0.18 6.6±0.7 6.2±0.11 6.0±0.41
0.7 18.6±3.6 18.9±3.2 18.5±0.26 19.3±3.15 6.7±0.09 6.4±0.25 6.5±0.14 6.6±0.74
1 19.1±0.25 19.3±4.5 19.5±0.14 19.4±2.15 6.6±0.63 6.6±0.14 6.3±0.06 6.1±0.21

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Table 6.27. Amount of IAA produced in μg/mL by Five promising bacteria of Citrus-based cropping
system with different concentration of Tryptophan.
Bacterial Conc. of *Amount of IAA Produced in μg /mL *Change of pH
isolate Tryptophan 24hrs 48hrs 72hrs 96hrs 24hrs 48hrs 72hrs 96hrs
g/L
CBC1 0 19.6±1.25 20.1±0.14 20.5±0.75 21.2±1.25 6.9±1.31 6.2±1.25 5.8±0.25 5.7±0.02
0.3 19.8±0.85 20.5±0.45 21.2±1.25 22.0±0.78 6.8±.1.21 6.5±2.36 6.1±0.36 5.3±0.14
0.7 20.9±0.75 21.6±0.32 21.7±1.56 22.5±0.85 6.9±1.25 6.2±2.44 5.8±0.11 5.1±0.36
1 18.6±0.83 18.5±0.41 18.2±0.85 18.3±1.24 6.7±1.45 5.8±0.46 5.6±0.14 5.1±0.14
CBC3 0 20.8±1.12 20.4±0.15 20.1±0.79 19.8±1.26 6.7±1.36 6.1±0.85 5.7±0.25 4.9±0.25
0.3 21.6±2.14 21.8±0.36 22.5±0.14 21.9±1.75 6.8±1.25 6.4±0.79 6.1±0.18 5.4±0.14
0.7 22.1±3.41 21.6±1.51 22.1±1.26 21.8±1.85 6.7±1.03 6.2±0.46 5.9±0.09 5.7±0.36
1 23.3±4.52 19.3±1.72 18.6±1.34 18.7±1.11 6.8±1.05 6.1±0.84 5.7±0.15 5.4±0.15
CBC6 0 22.2±0.85 22.1±1.36 21.8±2.34 21.6±0.84 6.9±1.25 6.3±0.36 5.8±0.11 5.1±0.39
0.3 23.6±1.64 24.4±1.8 24.7±3.15 23.8±0.75 6.8±0.98 6.1±0.14 5.8±0.32 5.3±0.25
0.7 22.2±1.35 20.1±1.25 19.6±4.26 19.3±0.65 6.7±0.45 6.2±0.15 5.6±0.18 5.1±0.85
1 20.1±1.35 19.5±0.89 19.4±1.28 19.1±0.47 6.8±0.35 6.4±0.32 6.1±0.17 5.3±0.74
CBC12 0 28.2±1.43 27.6±0.75 27.4±1.25 26.5±0.15 6.7±0.48 6.4±1.25 5.4±0.32 5.1±0.36
0.3 29.2±2.1 28.8±0.65 27.6±0.45 27.8±0.36 6.8±1.23 6.1±1.36 5.1±0.25 4.8±0.11
0.7 28.1±1.63 27.9±0.73 27.6±0.85 27.5±1.23 6.7±1.36 5.9±2.14 5.2±0.14 4.7±0.43
1 27.8±1.42 27.7±0.14 26.5±0.46 26.3±1.56 6.7±1.25 5.4±2.36 5.1±0.06 4.8±0.74
CBC13 0 24.4±1.31 24.8±1.28 24.8±1.74 23.7±0.18 6.6±0.48 6.2±0.85 5.7±0.08 5.2±0.23
0.3 23.5±0.85 23.6±2.14 24.9±1.56 24.4±1.84 6.6±0.46 6.3±0.46 6.4±0.45 6.1±0.85
0.7 26.3±0.79 24.1±2.14 23.8±2.11 23.6±1.46 6.7±0.36 6.4±0.14 5.8±0.63 5.3±0.45
1 25.2±0.71 24.8±1.25 23.2±2.36 22.8±1.32 6.8±0.85 6.1±0.17 5.7±0.87 4.9±0.36

Table 6.28. Amount of IAA produced in μg/mLby Five promising bacteria of Guava-based cropping
system with different concentration of Tryptophan

Bacterial Conc. of *Amount of IAA Produced inμg /mL *Change of pH

isolate Tryptophan 24hrs 48hrs 72hrs 96hrs 24hrs 48hrs 72hrs 96hrs
g/L
GBC1 0 15.2±1.21 18.6±1.32 19.3±1.24 20.1±0.87 6.7±0.12 6.3±0.14 5.8±0.13 5.1±0.11
0.3 16.2±1.23 18.6±1.25 19.7±1.25 20.7±1.24 6.8±0.11 6.1±0.25 5.8±0.12 4.7±0.16
0.7 18.2±0.89 19.2±1.32 20.4±2.34 22.3±1.23 6.7±0.11 6.2±0.12 5.4±0.31 4.8±0.84
1 18.1±1.5 18.3±1.4 18.7±1.26 18.2±1.25 6.8±0.42 6.5±0.15 6.5±0.16 6.2±0.16
GBC3 0 26.3±1.23 27.4±1.31 29.3±1.05 30.4±1.03 6.9±0.31 6.2±0.16 6.1±0.15 5.4±0.26
0.3 25.4±0.56 26.5±1.36 27.4±0.89 29.2±1.36 6.8±0.56 6.4±0.13 5.7±0.24 5.1±0.31
0.7 26.5±2.31 27.3±1.25 26.3±0.87 27.3±1.24 6.7±0.11 6.1±0.31 5.2±0.15 4.8±0.15
1 25.5±2.45 26.7±1.34 27.2±1.24 26.8±1.26 6.8±0.23 6.5±0.14 6.1±0.13 5.4±0.14
GBC12 0 30.12±1.45 32.25±1.20 34.26±1.75 36.32±0.89 6.7±0.11 5.2±0.12 4.8±0.74 4.6±0.25
0.3 32.24±1.56 31.9±1.25 34.56±1.23 35.25±0.48 6.8±0.32 5.4±0.38 4.8±0.45 4.2±0.89
0.7 33.45±1.36 33.1±1.36 33.2±0.89 33.4±0.75 6.7±0.14 5.8±0.16 5.1±0.61 4.6±0.56
1 32.5±1.24 33.2±1.47 33.48±0.87 33.7±1.25 6.8±0.25 6.5±0.15 6.2±0.31 5.8±0.16
GBC14 0 18.56±1.41 20.45±1.25 23.56±1.21 27.2±0.32 6.7±0.31 6.2±0.13 5.4±0.15 4.7±0.12
0.3 19.8±1.21 21.4±1.32 26.34±1.32 28.26±1.25 6.8±0.02 6.1±0.12 5.7±0.56 5.3±0.31
0.7 21.23±1.34 22.36±1.25 28.4±1.05 26.4±0.46 6.7±0.14 6.2±0.31 5.8±0.35 5.1±0.45
1 23.4±1.25 21.8±1.26 24.26±1.24 23.6±1.23 6.8±0.31 5.2±0.12 4.8±0.14 4.8±0.36
GBC16 0 20.45±1.34 23.56±1.85 26.45±1.4 28.4±0.98 6.9±0.13 6.7±0.16 5.8±0.85 4.2±0.15
0.3 21.4±1.25 24.2±1.26 26.7±1.25 27.2±0.48 6.8±0.14 6.4±0.32 6.3±0.23 6.1±0.18
0.7 23.5±1.05 24.3±1.24 26.4±1.32 28.1±0.46 6.7±0.32 5.8±0.21 4.9±0.52 4.7±0.14
1 24.1±1.11 25.3±1.34 27.2±1.05 29.4±0.87 6.8±0.15 6.2±0.22 5.2±0.36 4.8±0.74

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Table 6.29. Amount of IAA produced in μg/mLby Five promising bacteria of Dragon fruits-based
cropping system with different concentration of Tryptophan.

Bacterial Conc. of *Amount of IAA Produced in μg /mL *Change of pH

isolate Tryptophan 24hrs 48hrs 72hrs 96hrs 24hrs 48hrs 72hrs 96hrs
g/L
DBC5 0 12.5±1.25 14.2±1.31 16.2±1.25 18.2±1.25 6.9±0.15 6.1±0.45 5.7±0.14 4.8±0.28
0.3 14.2±1.32 16.2±1.32 17.2±1.78 19.2±1.35 6.8±0.46 6.2±0.12 5.8±0.63 4.9±0.26
0.7 14.8±1.26 17.2±1.25 18.2±1.59 20.1±2.34 6.7±0.15 5.8±0.32 5.9±0.52 4.8±0.36
1 15.2±1.34 17.9±1.24 18.6±1.59 21.2±3.15 6.8±0.42 6.7±0.25 6.4±0.41 6.2±0.45
DBC6 0 14.35±1.35 16.3±1.03 17.2±1.48 19.2±3.05 6.7±0.63 6.4±0.15 6.2±0.26 5.8±0.16
0.3 15.2±1.21 17.2±1.25 18.2±1.65 20.1±20.5 6.8±0.18 5.8±0.16 5.7±0.15 5.5±0.15
0.7 16.3±1.11 17.5±1.36 19.3±1.45 21.3±0.86 6.8±0.75 6.1±0.45 5.8±0.31 4.9±0.19
1 18.2±1.21 20.1±1.24 20.5±1.65 21.2±0.75 6.7±0.62 6.1±0.85 5.7±0.85 4.9±0.48
DBC7 0 11.25±1.31 12.3±1.34 14.5±1.28 16.3±0.45 6.8±0.45 5.8±0.64 5.2±0.82 4.8±0.44
0.3 13.85±1.32 16.3±1.75 17.2±1.34 18.5±1.26 6.7±0.49 6.2±0.75 6.0±0.15 5.8±0.15
0.7 14.36±1.21 16.3±1.21 18.5±1.75 20.1±2.31 6.8±0.14 6.5±0.65 6.1±0.26 5.9±0.23
1 15.3±1.34 16.2±2.13 18.2±1.25 20.6±1.25 6.7±0.63 6.3±0.26 5.7±0.14 5.1±0.16
DBC11 0 17.5±1.36 19.5±2.15 20.1±0.89 23.5±3.05 6.8±0.85 6.5±0.32 5.7±0.23 5.1±0.47
0.3 18.2±1.25 20.4±2.34 22.4±0.75 24.2±1.26 6.7±0.56 6.1±0.45 6.0±0.25 5.8±0.15
0.7 19.3±1.21 20.4±1.25 21.5±0.45 24.6±3.21 6.8±0.15 5.8±0.86 5.7±0.85 5.1±0.62
1 20.3±1.31 22.2±0.89 23.5±1.15 25.3±1.56 6.8±0.48 6.2±0.85 6.0±0.16 5.5±0.18
DBC14 0 18.6±1.21 20.5±1.24 21.3±1.28 22.5±2.13 6.7±0.36 5.8±0.15 4.8±0.14 4.7±0.46
0.3 20.4±1.35 21.3±2.15 22.6±1.35 23.5±1.15 6.7±0.35 5.2±0.62 4.3±0.32 4.2±0.16
0.7 21.4±0.89 22.5±2.35 23.4±0.56 25.6±2.18 6.8±0.85 5.4±0.85 5.1±0.22 4.9±0.16
1 22.4±0.87 22.8±0.78 23.4±0.48 22.6±2.89 6.8±0.45 6.1±0.63 6.3±0.18 5.8±0.32

6.3.9.9.1. PCR amplification of Isolated bacterial genomic DNA

PCR amplification is a technique employed to examine the genomic DNA of bacteria
in different crop diversification practices. The process entails preparing a reaction
mixture including precise primers, DNA polymerase, and template DNA. The
procedure involves denaturation, primer annealing, and extension cycles in a thermal
cycler. Efficient amplification relies on the use of optimal cycle conditions (Garibyan
et al.2013). Following the PCR process, gel electrophoresis is employed to validate
the successful amplification, so guaranteeing sterility. This technique facilitates the
examination of various bacterial genomes present in agricultural systems, enabling the
selective amplification of certain DNA sections for further analysis and investigation.
The PCR reaction is confirmed to be specific by successfully amplifying a single band
at the 1.5 kb point of the DNA ladder on the agarose gel. A clear band at the
anticipated size confirms precise amplification of the specific DNA segment. Table
6.30. represent the detail description of reaction information, sample information and
primer information for the PCR amplification.

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Table 6.30. Detailed genomic description of bacterial DNA isolation, including the process of PCR amplification and the diverse agricultural system.

Reaction Information Sample Information Primer Information

Diversified Sequencing
cropping Primer
Sample Sample Sample Product Primer
system Plasmid/PCR 260/280 Primer Sequence (5 to 3) Conc
Name Forward* Reverse* Conc. Volume Size(bp) Name**
(pmol/µl)
(ng/µl)
Banana PCA 23 F NA PCR 75.5 1.822 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
based PCA 76 F NA PCR 64.8 1.811 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
cropping PCA 86 F NA PCR 58.5 1.75 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
system PCA 101 F NA PCR 52.6 1.83 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
PCA 6 F NA PCR 79.3 1.78 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
Citrus PCA 7 F NA PCR 62.12 1.79 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
based
PCA 11 F NA PCR 70.12 1.74 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
cropping
system PCA 28 F NA PCR 57.25 1.86 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
PCA 55 F NA PCR 68.52 1.83 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
Guava PCA 4 F NA PCR 58.56 1.74 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
based PCA 15 F NA PCR 82.12 1.79 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
cropping PCA 31 F NA PCR 55.12 1.82 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
system PCA 37 F NA PCR 77.12 1.81 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
PCA 14 F NA PCR 95.26 1.84 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
Dreagan
fruits- PCA 15 F NA PCR 82.12 1.81 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
based PCA 16 F NA PCR 91.12 1.78 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
cropping PCA 17 F NA PCR 74.26 1.75 25 1495 27F AGAGTTTGATCCTGGCTCAG 10
system
PCA 18 F NA PCR 92.12 1.82 25 1495 27F AGAGTTTGATCCTGGCTCAG 10

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6.3.9.9.2. Identification of bacterial isolates based on sequence homology

The NCBI database was utilized to examine nucleotide sequences obtained from isolated
bacteria in order to identify any sequence similarity. The BLAST tool was employed to
compare the isolated bacteria's sequences with the available 16SrRNA sequences in order
to ascertain the degree of similarity between them and the recorded sequences in the
database. This process facilitates the identification and classification of bacterial species by
analyzing the level of similarity seen in their genomic sequences. BLASTn, also known as
Basic Local Alignment Search Tool for nucleotides, compares the genetic sequence of
interest with a large collection of known sequences in the NCBI database (Jhuma et al.
2021). This comparison facilitates scientists in assessing the extent of resemblance
between the isolated bacterial DNA and established sequences, hence assisting in the
identification and categorization of the bacterial species or genus. Utilizing the extensive
genomic data accessible in public databases such as NCBI, this approach plays a vital role
in describing and categorizing recently identified bacterial species or strains (O'Leary et al.
2016). The study discovered four possible bacterial species, including Bacillus
bingmayongensis strain PCA86, Bacillus sp. strain PCA101, Fredinandcohnia onubensis
strain PCA23, and Staphylococcus saprophyticus strain PCA76, in a banana-based
cropping system. These species were identified using the NCBI database. In addition,
within the Citrus-based cropping system, five potential PGPR strains have been identified.
These strains are Rossellomore avietnamensis strain PCA6, Bacillus mojavensis strain
PCA7, Fredinandcohnia humi strain PCA11, Bacillus subtilis strain PCA55, and
Staphylococcus equorum subsp. linens strain PCA28. The identification process was
carried out using the NCBI database analysis. Moreover, in a Guava based cropping system
using four possible strains of PGPR were identified. These strains were Bacillus sp. strain
PCA37, Staphylococcus succinus strain PCA4, Staphylococcus equorum strain PCA15,
and Staphylococcus succinus subsp. succinus strain PCA31. The identification was done by
NCBI based BLAST analysis. However, in a cropping system based on Dragon-fruits, the
identification of potent PGPR strains such as Priestia aryabhattai strain PCA 51,
Brachybacterium ginsengisoli strain PCA69, Staphylococcus cohnii strain PCA72, Bacillus
halotolerans strain PCA75, and Brevibacterium frigoritolerans strain PCA78 is done
through NCBI based BLAST analysis. The detail description of bacterial isolate, accession
number, percentage identification, accession length is mention in Table 6.31. The sequence
similarity of all selected PGPR strain from diversified cropping system are represented in
Table 6.32.

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Table 6.31. List of potential PGPR strains, selected from the diversified cropping system including their strain and similarity isolate names, Percentage Identification,NCBI

Serial No Name of Bacterial Species Accession Percentage Max Total Query Accession
Number Identification score score cover Length
Banana Based Cropping Bacillus bingmayongensis strain PCA86 OK087331 98.56% 1354 1354 97% 1443
System
Bacillus sp. strain PCA101 OK090423 97.22% 1158 1158 100% 1472
Fredinandcohnia onubensis strain PCA23 OK090421 97% 1267 1267 97% 1429
Staphylococcus saprophyticus strain PCA76 OK090433 99% 1690 1690 98.44% 1553
Citrus Based Cropping Rossellomorea vietnamensis strain PCA6 OK090424 82.01% 817 817 98% 1388
System
Bacillus mojavensis strain PCA7 OK090420 99.13% 1853 1853 98% 1526
Fredinandcohnia humi strain PCA11 OK090419 99.61% 1869 1869 99% 1429
Bacillus subtilis strain PCA55 OK090422 99.71% 1249 1249 100% 1550
Staphylococcus equorum subsp. linens strain OK090432 99% 1000 1000 98.93% 1535
PCA28
Guava Based Cropping Bacillus sp. strain PCA37 OK087329 97.85% 1279 1279 99% 1465
System
Staphylococcus succinus strain PCA4 OK090434 95.36% 1844 1844 90% 1548
Staphylococcus equorum strain PCA15 OK090428 97% 1201 1201 97.33% 1494
Staphylococcus succinus subsp. succinus OK090435 97.89% 99% 1548
902 902
strain PCA31
Drogan fruits Priestia aryabhattai strain PCA 51 OK087330 98.37% 1938 1938 99% 1533
Based Cropping System
Brachybacterium ginsengisoli strain PCA69 OK090425 99.88% 1513 1513 97% 1416
Staphylococcus cohnii strain PCA72 OK090427 97.95% 1092 1092 97% 1535
Bacillus halotolerans strain PCA75 OK090418 97.43% 1714 1714 96% 1468
Brevibacterium frigoritolerans strain PCA78 OK090426 99.27% 1234 1234 99.27% 1503
accession number in details.

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Table 6.32. NCBI based BLAST Sequence similarity of all PGPR strain from diversified cropping system.
Bacillus bingmayongensis NR_148248.1 Bacillus bingmayongensis strain FJAT-13831 98.65%
PCA 31 NR_113991.1 Bacillus pseudomycoides strain NBRC 101232 98.07%
NR_115526.1 Bacillus cereus strain IAM 12605 97.92%
NR_157736.1 Bacillus tropicus strain MCCC 1A01406 97.92%
NR_157732.1 Bacillus nitratireducens strain MCCC 1A00732 97.92%
Bacillus humi PCA19 NR_025626.1 Bacillus humi strain LMG 22167 99.61%
NR_125590.1 Bacillus massiliosenegalensis strain JC6 98.41%
NR_135903.1 Bacillus wuyishanensis strain FJAT-17212 97.55%
NR_042286.1 Bacillus herbersteinensis strain D-1,5 97.79%
NR_149252.1 Bacillus onubensis strain 0911MAR22V3 97.88%
Bacillus megaterium NR_164882.1 Bacillus zanthoxyli strain 1433 100%
PCA23 NR_112636.1 Bacillus megaterium NBRC 1530 99.70%
NR_133978.1 Bacillus qingshengii strain G19 99.10%
NR_118382.1 Bacillus flexus strain SBMP3 98.64%
Bacillus onubensis PCA11 NR_149252.1 Bacillus onubensis strain 0911MAR22V3 99.64%
NR_133024.1 Bacillus timonensis strain 10403023 97.11%
NR_025626.1 Bacillus humi strain LMG 22167 97.11
NR_147383.1 Bacillus sinesaloumensis strain Marseille-P3516 96.76%
Bacillus onubensis PCA17 NR_149252.1 Bacillus onubensis strain 0911MAR22V3 99.86%
NR_133024.1 Bacillus timonensis strain 10403023 97.9%
NR_025626.1 Bacillus humi strain LMG 22167 97.9%
NR_147383.1 Bacillus sinesaloumensis strain Marseille-P3516 97.63%
Bacillus simplex PCA25 NR_042136.1 Bacillus simplex NBRC 15720 = DSM 1321 99.83%
NR_117474.1 Brevibacterium frigoritolerans strain DSM 8801 99.83%
NR_042083.1 Bacillus muralis strain LMG 20238 99.66%
NR_044170.1 Bacillus butanolivorans strain K9 98.82%
Bacillus subtilis PCA 27 NR_112116.2 Bacillus subtilis strain IAM 12118 99.71%
NR_104873.1 Bacillus subtilis subsp. inaquosorum strain BGSC 3A28 99.56%
NR_024693.1 Bacillus mojavensis strain IFO15718 99.42%
NR_115931.1 Bacillus halotolerans strain LMG 22477 99.27%
NR_104919.1 Bacillus tequilensis strain 10b 99.42%
Bacillus subtilis PCA13 NR_024931.1 Bacillus subtilis subsp. spizizenii strain NRRL B-23049 99.88%
NR_104873.1 Bacillus subtilis subsp. inaquosorum strain BGSC 3A28 99.77%
NR_115931.1 Bacillus halotolerans strain LMG 22477 16S 99.77%
NR_112116.2 Bacillus subtilis strain IAM 12118 99.65%
NR_024693.1 Bacillus mojavensis strain IFO15718 99.65%
Bacillus subtilis subsp. NR_104873.1 Bacillus subtilis subsp. inaquosorum 99.84%
Inaquosorum PCA21 NR_024931.1 Bacillus subtilis subsp. spizizenii strain NRRL B-23049 99.68%
NR_115931.1 Bacillus halotolerans strain LMG 22477 99.52%
NR_151897.1 Bacillus nakamurai strain NRRL B-41091 16S 99.36
Brachybacterium sp. NR_133984.1 Brachybacterium ginsengisoli strain DCY80 99.61
PCA29 NR_169311.1 Brachybacterium vulturis strain VM2412 98.35%
NR_169313.1 Brachybacterium avium strain VR2415 97.86%
Pseudarthrobacter NR_108849.1 Pseudarthrobacter siccitolerans strain 4J27 98.84%
siccitolerans NR_026191.1 Arthrobacter pascens strain DSM 20545 98.26
PCA33 NR_041545.1 Arthrobacter oryzae strain KV-651 98.26%
NR_042573.1 Pseudarthrobacter defluvii strain 4C1-a 97.96%
NR_041400.1 Pseudarthrobacter niigatensis strain LC4 97.82%
Staphylococcus arlettae NR_041926.1 Staphylococcus equorum subsp. linens strain RP29 99.58%
PCA 35 NR_156818.1 Staphylococcus edaphicus strain CCM 8730 97.50%
NR_024664.1 Staphylococcus arlettae strain ATCC 43957 97.29%
Staphylococcus NR_041926 Staphylococcus equorum subsp. linens strain RP29 99.11%
equorumPCA2 NR_027520.1 Staphylococcus equorum strain PA 231 99.11%
NR_113350.1 Staphylococcus xylosus strain JCM 2418 97.32%
NR_037046.1 Staphylococcus cohnii subsp. urealyticus strain CK27 97.32%
Staphylococcus equorum NR_041926.1 Staphylococcus equorum subsp. linens strain RP29 99.13%
subsp. Linens PCA5 NR_027520.1 Staphylococcus equorum strain PA 231 16S 99.13%
NR_037046.1 Staphylococcus cohnii subsp. urealyticus strain CK27 96.94%
Staphylococcus NR_074999.2 Staphylococcus saprophyticus subsp. ATCC 15305 97.95%
saprophyticus PCA15 NR_036902.1 Staphylococcus cohnii strain GH 137 97.55%
NR_156818.1 Staphylococcus edaphicus strain CCM 8730 97.98%
Staphylococcus Sp. NR_027520 Staphylococcus equorum strain PA 231 100%
PCA7 NR_113350.1:29-618 Staphylococcus xylosus strain JCM 2418 98.31%
NR_028667.1 Staphylococcus succinus subsp. succinus strain AMG-D1 98.31%
NR_036902.1 Staphylococcus cohnii strain GH 137 98.14

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6.3.9.9.3. Phylogenetic analysis

The study utilized ClustalW v1.8 to align biological sequences for the purpose of
comparison, resulting in the creation of a phylogenetic dendrogram to visually
represent evolutionary connections. A study using bootstrap method was conducted on
1000 datasets in MEGA X to evaluate the reliability of the tree, by examining the
similarities and differences across sequences. This technique deduced evolutionary
relationships and verified their certainty using statistical validation, offering insights
into genetic evolution inside biological data. The utilization of ClustalW and MEGA
X facilitated the deduction of evolutionary connections and the verification of their
certainty. A tree was constructed using bootstrap percentages obtained from 1,000
replications (Challa et al. 2019). The bar on the tree shows the estimated number of
nucleotide substitutions per nucleotide, indicating the level of genetic diversity
between the sequences. The analysis in the research utilized MEGA X software with
1,000 bootstrap repetitions (Eshaghi et al., 2012). The Figure 6 displays the
phylogenetic tree of the chosen PGPR strains from various cropping systems. The
phylogenetic tree was generated using the neighbor-joining approach, using the
sequence similarity of all the possible PGPR strains and their closely related
phylogenetic counterparts as seen in Figure 7. In addition, a phylogenetic tree was
created for PGPR that were obtained from a banana-based cropping system. The
neighbor-joining approach was used, and the sequence similarity of all the chosen
PGPR strains was considered. The resulting phylogenetic tree, which includes the
closest relatives of the selected strains, is depicted in Figure 8. Nevertheless, the
neighbor-joining approach was used to build the phylogenetic tree for the PGPR
isolated from the citrus-based cropping system, and Figure 9 representation of the
closed phylogenetic relationship and all of the probable PGPR strains that were
chosen had comparable sequences. Additionally, the neighbor-joining approach was
used to create the phylogenetic tree for the PGPR isolate from the Guava-based
cropping system. Figure 10 shows the closed phylogenetic relative of each probable
PGPR strain that was chosen and their sequence similarity. Furthermore, using the
neighbor-joining approach and the sequence similarity of every probable PGPR strain
that was chosen, together with their closed phylogenetic relative shown in Figure 11, a
phylogenetic tree was built for PGPR isolated from dragon fruit-based cropping
system. However, the investigation of prospective rhizobacteria that promote plant
development, known as plant-growth promoting rhizobacteria (PGPR), entails
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evaluating their ability to build biofilms using scanning electron microscopy (SEM),
as shown in Figures 4 and 5. This technique offers precise visualization of biofilm
formations, allowing for the observation of bacterial organization, extracellular
chemicals, and interconnections. This knowledge facilitates comprehension of the
activity of PGPR in the rhizosphere, hence augmenting their utilization for promoting
plant development and health in agriculture. Scanning electron microscopy (SEM)
examination offers in-depth understanding of the process of biofilm formation by
PGPR strains, shedding light on their ability to colonize and promote plant
development in agricultural environments. Gaining insight into the architecture of
biofilms facilitates the formulation of tactics to combat biofilm-associated infections,
enhance efficiency in industrial operations, and use advantageous biofilms for
medical, agricultural, and environmental purposes.

Biofilm formation of Biofilm formation of

Bacillus bingmayongensis strain PCA 86 Fredinandcohnia onubensis strain
PCA23

Biofilm formation of Biofilm formation of

Rossellomorea svietnamensis strain Bacillus mojavensis strain PCA7
PCA6
Figure 6.4.The evaluation of PGPR biofilm formation includes selecting, cultivating, and measuring on
surfaces, facilitated by SEM for detailed visualization, enhancing agricultural outcomes.

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Biofilm formation of Biofilm formation of

Bacillus sp. strain PCA37 Staphylococcus succinus strain PCA4

Biofilm formation of Biofilm formation of

Priestia aryabhattai strain PCA 51 Brevibacterium frigoritolerans strain
PCA78zzz
Figure 6.5. SEM analysis reveals PGPR biofilm structure, aiding comprehension of colonization
capacity and its role in promoting plant growth in agriculture.

Figure 6.6. The study investigated the 16S rDNA sequences from potential PGPR strains from
diversified cropping systems using MEGA X, a phylogenetic analysis tool. A tree was built using
bootstrap percentages from 1,000 replications.

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Figure 6.7. Comparing 16S rDNA sequences of PGPR isolates with relatives generated a phylogenetic
tree using neighbor-joining, with bootstrap values. Substitution rates indicate genetic differences.

.
Figure 6.8. Phylogenetic tree of banana-based PGPR isolates and relatives made with neighbor-joining
algorithm, showing branch support and nucleotide substitution rates.

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Figure 6.9. A phylogenetic tree compares 16S rDNA sequences of Citrus-based PGPR isolates and
relatives, with branch support and nucleotide substitution rates.

Figure 6.10. Comparing 16S rDNA sequences of Guava-based PGPR isolates and relatives created
a phylogenetic tree, displaying branch support and nucleotide substitutions.

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Figure 6.11. A phylogenetic tree, comparing 16S rDNA of Dragon-fruit-based PGPR isolates with
relatives, depicts branch support and nucleotide substitution rates.

6.4. Conclusion
The study identified variations in the reactivity of rhizobacteria based on their
morphology and pH levels across different agricultural systems. This study
demonstrates the alterations in microbial behavior under agricultural conditions.
Comprehending the complex interplay between rhizobacteria and the environment is
essential for sustainable farming. An understanding of how these bacteria respond to
pH levels enables researchers to enhance crop growth and improve soil quality. By
utilizing this knowledge, farmers may effectively utilize microbial dynamics to
enhance the availability of nutrients in an environmentally friendly way and promote
the overall health of the ecosystem. The research emphasizes the need to consider
microbial interactions in various agricultural settings to promote knowledgeable,
adaptable, and environmentally aware farming practices. The study revealed that
rhizobacteria exhibit a high degree of tolerance towards salt and diverse temperature
conditions. These isolates exhibited the production of indole-3-acetic acid (IAA),
enzymes that solubilize phosphate, hydrogen cyanide (HCN), and siderophores. These
compounds enhance soil quality, promote plant growth, and facilitate nutrient
absorption. Isolates of PGPR obtained from farms enhanced nutritional quality. The
high generation of siderophores in multiple systems demonstrated the potential of 13-
15 isolates in diverse cropping system. Certain varieties of bananas and citrus fruits

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exhibited enhanced ammonia production for nitrogen conversion. The phosphate

solubilization demonstrated exceptional strains of banana, citrus, guava, and dragon
fruit. Rhizobacteria enhance nutrient accessibility and promote plant growth in
diverse agricultural settings, indicating the potential for sustainable farming practices.
The study demonstrates that rhizobacteria enhance nutrient availability in various
agricultural environments. The bio-dissolution of zinc and solubilization of potassium
exhibited distinct patterns, with specific strains demonstrating superior performance.
The concentration of tricalcium phosphate (TCP) and the duration of incubation had
an impact on the ability of bacterial strains to solubilize phosphate and produce
indole-3-acetic acid (IAA) in citrus, guava, and dragon fruit systems. The impact of
TCP concentrations (ranging from 2.5 to 10.0 g/L) and incubation durations (ranging
from 24 to 120 hours) varied among various strains. The combination of citrus, guava,
and dragon fruit was successful. The synthesis of IAA exhibited variation in response
to different dosages of tryptophan and over incubation periods ranging from 24 to 96
hours. The concentration of citrus IAA produced by CBC12 was higher than that
produced by BBC7 in bananas. The data presented indicate that rhizobacterial
reactions to nutrient solubilization and hormone synthesis vary among agricultural
systems, implying their potential for promoting sustainable agriculture. The study
molecularly characterized Plant Growth-Promoting Rhizobacteria (PGPR) from
various cropping regimes. The recovered bacteria's unique DNA segments were
confirmed using gel electrophoresis and PCR. The BLAST testing revealed the
presence of Bacillus, Staphylococcus, and Fredinandcohnia PGPR strains in banana,
citrus, guava, and dragon fruit. The phylogenetic investigation revealed the diversity
of these strains and their dendrogram connections to closely related strains. However,
scanning electron microscopy (SEM) was used to analyze the colonization patterns in
the biofilms formed by various strains of plant growth-promoting rhizobacteria
(PGPR). The comprehensive study of chemicals and genes associated with biofilms
shown their capacity to enhance agricultural plant development and overall health.
The genetic connections and ability to build biofilms demonstrate how these strains of
PGPR can be utilized to enhance both crop yield and environmental sustainability.
The significance of this comprehensive investigation on Plant Growth-Promoting
Rhizobacteria (PGPR) across many agricultural settings cannot be overstated. The
study demonstrates the adaptive mechanisms of rhizobacteria to diverse settings,
which subsequently impact nutrient accessibility and the overall well-being of
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ecosystems. Microbial reactions aid in optimizing agricultural practices according to

pH levels, enhancing crop growth, and promoting soil well-being. Furthermore,
Rhizobacteria significantly influence plant growth, nitrogen absorption, and soil
conditions through the production of growth-stimulating compounds. The molecular
identification and characterization of PGPR strains unveil their genetic diversity,
evolutionary connections, and ability to produce biofilms, allowing for targeted
agricultural applications. This study establishes the foundation for intelligent and
sustainable farming methods that utilize microbial interactions to enhance agricultural
productivity and safeguard the environment.

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