From Sea to Soil: Marine Bacillus subtilis enhancing chickpea production through in vitro and in vivo plant growth promoting traits

Abstract

Various strategies are used to augment agricultural output in response to the escalating food requirements stemming from population expansion. Out of various strategies, the use of plant growth-promoting bacteria (PGPB) has shown promise as a viable technique in implementing new agricultural practices. The study of PGPB derived from rhizospheric soil is extensive, but there is a need for more exploration of marine microorganisms. The present research aims to investigate the potential of marine microorganisms as promoters of plant growth. The marine microbe Bacillus subtilis used in current study has been discovered as a possible plant growth-promoting bacterium (PGPB) as it showed ability to produce ammonia, solubilize potassium and phosphate, and was able to colonize chickpea roots. Bacillus subtilis exhibited a 40% augmentation in germination. A talc-based bio-formulation was prepared using Bacillus subtilis, and pot experiment was done under two conditions: control (T1) and Bacillus treated (T2). In the pot experiment, the plant weight with Bacillus treatment increased by 14.17%, while the plant height increased by 13.71% as compared to control. It also enhanced the chlorophyll content of chickpea and had a beneficial influence on stress indicators. Furthermore, it was noted that it enhanced the levels of nitrogen, potassium, and phosphate in the soil improving soil quality. The findings showed that B. subtilis functioned as a plant growth-promoting bacteria (PGPB) to enhance the overall development of chickpea.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01238-1.

Introduction

Consequences of the global increase in human population and the resulting environmental degradation include the possibility that global food production may soon be insufficient to feed everyone on the planet. Therefore, boosting farming production in the coming decades is essential. One of the most well-studied methods for encouraging plant development is by supplying plants the nutrients they lack, like nitrogen and essential minerals like potassium and phosphorus with help of microbes [1]. Many agricultural soils are deficient in one or more of these nutrients or only contain them in plant insoluble forms [2, 3]. Hence, not providing plants sufficient nutrients for its optimal growth. For the same purpose, a variety of chemical fertilisers and biological fertilisers are utilised. Since chemical fertilisers were discovered to be hazardous to the environment the use of biological fertilisers has become growing in significance [4]. Use of biological fertilizers includes both microbes and plant extract [5, 6]. Microorganisms are chosen over plant extracts as biological fertilisers because they are easier to cultivate, more readily available, and more effective. Plant growth promoting bacteria (PGPB) represent a potential remedy to this issue. Normally, all plants release a fluid that is rich in carbon and nourishes the bacteria whereas microbes increase the availability of nutrients and decrease abiotic stressors such an excess or shortage of water or salts [7]. In the agro-farming system, PGPB can be successfully regulated as an alternative strategy to raise their yields, and to result in the least usage of synthetic fertilisers [8, 9]. Plant growth-promoting bacteria (PGPB) are microorganisms that play a significant part in plant growth [10, 11]. The primary roles of PGPB include the production of plant hormones and improvement of nutrient supply to plants [12, 13]. Such microorganisms are also beneficial for restoring the soil's fertility [14].

Various species of bacteria have been recognised as plant growth-promoting bacteria, with Bacillus and Pseudomonas spp. being the most prevalent. One of the most often utilised and investigated PGPB is Bacillus species. They are non-pathogenic and highly promising contender for agricultural uses due to its capability of produces endospores which are very resilient to a variety of abiotic stressors, such as drought, temperature, or nutrient constraint [15, 16]. Additionally, it is known that certain Bacillus species can fix atmospheric nitrogen, encourage the nodulation of other bacteria, and hence enhance the colonisation of local, symbiotic rhizobacteria [17]. Plant growth can be actively influenced by Bacillus, which encourages cell proliferation and seed germination either by synthesizing certain compounds directly or by triggering the release in plants by indirectly secreted molecules. In addition to growth promotion Bacillus aids plants in enduring environmental stress. According to reports, Bacillus alters the expression of plant genes to increase plant tolerance to salt and drought stress [18] proving as a vital bacterial for plant growth and development. The rhizospheric Bacillus, which are native to plants, are commonly used to make biofertilizer to help plants. However, we formulated a hypothesis to investigate the potential of marine Bacillus for growth promotion as this aspect of marine microbes needs to be more explored.

Present work focusses on boosting chickpea production as the legume chickpea has a significant nutritious function in the diets of many people in impoverished nations [19]. The uniqueness of chickpea lies in its remarkable protein concentration, which constitutes from 17 to 22% [20]. Furthermore, the chickpea crop has considerable health advantages, including as mitigating the risks of cardiovascular diseases, diabetes, and cancer [21]. Chickpeas are versatile crops that may be eaten directly as food or in many processed forms. They are also used as feed in various agricultural methods [22] and in countries like India its processed form is used in traditional skincare routine. Chickpea is a crucial crop cultivated in the highlands of several nations worldwide, serving as a significant source of income and food security. Chickpea production is the third highest globally, with an average annual output of 11.5 million tonnes, with the majority produced in India [21]. Chickpea is responsible for about half of India's pulse output, making it a significant and valuable crop for both its nutritional and economic benefits [23]. This work focuses on investigating marine Bacillus for its capacity to promote chickpea growth both in vitro as well as in vivo, as pot study is vital to check effectiveness of culture in nature environment.

Materials and methods

Isolation and Identification of organism

Organism was isolated from Gulf of Khambhat, Gujarat, India (22°30´N,72°61´E). Biochemical tests of Bergey’s Manual of Systematic Bacteriology were performed as described by Shi et. al., 2012. All the reagents used were obtained from Himedia (India). Sugar utilization was done with help of Himedia HiCarbo™ Kit. Fresh culture was sent to Eurofins for 16 s rDNA sequencing.

Optimization of growth

Organism was grown in marine broth (MB), nutrient broth (NB) and luria bertani broth (LB), different concentrations of salt, pH, temperature, and aeration. Its optical density was measured with UV–Visible spectrophotometer (Shimadzu 1800) till it reached its decline phase.

Indole acetic acid (IAA) production

Isolate was inoculated in nutrient broth (Himedia) supplemented with 200 µg/mL L- tryptophan (Himedia). The flasks were incubated at 27 °C for 3 days. After 3 days, 1 mL of the broth was centrifuged at 2700 X g for 15 min. This supernatant was used for measurement of IAA. Equal volume (1:1) of sample supernatant and Salkowski reagent were mixed. Salkowski’s reagent was prepared by mixing 50 mL 35% perchloric acid and 1 mL 0.5 M FeCl₃ solution. Development of pink colour indicates presence of IAA. For its quantitative measurement optical density was checked at 530 nm using UV–Visible spectrophotometer (Shimadzu UV-1800) [24].

Gibberellin production

The isolate was inoculated in 100 mL nutrient broth and incubated at 30 °C for 7 days. For 10 min, culture was centrifuged at 2600 X g. Gibberellin was detected using supernatant. To 15 mL supernatant, 2 mL zinc acetate (21.9 gm zinc acetate + 1 mL glacial acetic acid and volume was made up to 100 mL with distilled water) was added and left to sit at room temperature for 2 min. To this, 2 mL of 10.6% potassium ferrocyanide was added, and centrifuged for 10 min at 2600 X g. The supernatant was collected, equal volumes of 30% HCl was added, and the mixture was incubated for 75 min at RT. Greenish colour developed was measured at 254 nm using Shimadzu UV-1800 UV Visible spectrophotometer following incubation [25]. Gibberellin (HiMedia) was used as standard in the range 1–10 µg/mL.

Phosphate solubilization

Isolate was spot inoculated on Pikovskaya’s agar (Himedia) amended with 0.05% bromophenol blue (Himedia) and was incubated at 27 °C for 5 days. Zone of solubilization and colony diameter were measured for calculating solubilization index. For its quantitative estimation, isolate was inoculated in Pikovskaya’s broth (Himedia) and incubated at 27 °C for 5 days. One mL of broth was centrifuged at 8000 rpm for 10 min. This supernatant was used for estimation of phosphate solubilization using stannous chloride method. To 0.1 mL supernatant, 0.9 mL distilled water and 1 ml chloromolybdic acid (15 gm ammonium molybdate was added in 400 mL distilled water. To this, 400 mL 10 N HCl was added and final volume was made upto 1 L by distilled water) was added. In this mixture, 0.25 ml chlorostannous acid (2.5 gm stannous chloride was added in 10 mL HCl and final volume of 100 mL was made up with distilled water) was added and mixed which lead to development of blue colour. Final volume of 5 ml was made up with distilled water. Absorbance was measured at 600 nm using Shimadzu UV-1800 UV–Visible spectrophotometer [26]. Monobasic potassium phosphate was used as standard (HiMedia) ranging from 10–100 µg/mL.

Ammonia production

BS 90 culture was inoculated in peptone water broth (Himedia) and incubated for 5 days at 27 °C. One mL of culture was centrifuged at 2700 g for 10 min. Amount of ammonia produced was estimated using Nessler’s reagent. One mL Nessler’s reagent (Himedia) was added to 200 µL of supernatant and 8.5 mL of autoclaved distilled water was added to this. Development of brown colour indicated presence of ammonia. Its optical density was measured at 450 nm using UV–Visible spectrophotometer (Shimadzu 1800) [27] Standard ammonia was prepared using ammonium sulphate (0.1–1 µmol/mL).

Nitrogen fixing test

N-free Jensen media was used to detect nitrogen fixing ability of isolate. Isolate was streaked on Jensen media (Sucrose 20 gm, Dipotassium phosphate 1 gm, Magnesium sulphate 0.5 gm, Sodium chloride 0.5 gm, Ferrous sulphate 0.1 gm, Sodium molybdate 0.005 gm, Calcium carbonate 2 gm, Agar 15 gm per 1000 mL distilled water) and incubated at 27 °C for 2 days. Growth in plates is considered positive for nitrogen fixation [28].

Potassium solubilization

Isolate was spot inoculated on Alaksandrov’s medium (Himedia) amended with 0.05 g/mL of bromothymol blue (pH 7.2 ± 2) and incubated at 27 °C for 5 days. Colony diameter and solubilization zone was measured for calculating solubilization index. Potassium solubilization was measured quantitatively by inoculating 24 h old culture in Alaksandrov’s medium having potassium aluminosilicate as the only potassium source and incubated at 27 °C for 7 days. After 7 days, 1 mL of broth was centrifuged at 8000 rpm for 10 min and supernatant was used further. One mL 1 M sodium cobaltinitrite was added to 1 mL supernatant and incubated at 37 °C for 40 min, centrifuged at 8000 rpm for 10 min. Ten mL concentrated HCl was added to the pellet resulting in development of blue to green colour. Optical density was measured at 623 nm using UV–Visible spectrophotometer (Shimadzu 1800) [29]. Standard potassium chloride graph was prepared in the range of 10–100 mg/mL.

Zinc solubilization

Isolate was spot inoculated on nutrient agar (Himedia) containing 0.1% insoluble zinc oxide [30] and plates were incubated at 27 °C for 3 days. Solubilization index was calculated using the following formula:

$$Solubilizationindex=\frac{Zoneofsolubilization(cm)}{Colonydiameter(cm)}\times 100$$

Siderophore production

MM9 media was first defferated before use. For 100 ml MM9 minimal media, 0.1 M Tris–HCl was added to adjust pH 6.8. In the above media, 100 ml 0.25% 8-hydroxyquinoline was added. This mixture was transferred to separating funnel and mixed. Chloroform layer was discarded twice. The defferated media left in separating funnel was collected and used for qualitative estimation. Defferated media and CAS indicator was autoclaved separately and after autoclaving 10% CAS indicator was mixed with media and plates were poured. Isolate was inoculated on CAS blue agar plates for five days. Production of orange halos indicates siderophore production [31].

HCN production

For detection of HCN producers, isolate was streaked on nutrient agar plates supplemented with 4% w/v glycine (Himedia). Whatman filter paper (no. 3) soaked in 2% sodium carbonate (dissolved in 0.5% picric acid) was placed on the lid. Plates were sealed with parafilm and incubated in upright position for 5 days at 27 °C. Change in colour of filter paper from orange to brown indicated HCN production [32].

Water agar test

BS 90 was spread on water agar plates (1% w/v) and incubated for 7 days. After 7 days, 5 healthy chickpea seeds were selected randomly and surface sterilized (by dipping in distilled water for 1 min. followed by 70% methanol for 1 min. and rinsed with distilled water for 30 s) and were placed on water agar plate with BS 90 and without BS 90 (Control). Plates were incubated for 5 days and its germination was observed [33]. Germination percentage and vigor index were calculated by following formula:

$$\begin{array}{c}\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\%=\mathrm{Number}\mathrm{of}\mathrm{seeds}\mathrm{germinated}*100/\mathrm{Total}\mathrm{number}\mathrm{of}\mathrm{seeds}\hfill \\ \mathrm{Vigor}\mathrm{index}=\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\%*\mathrm{average}\mathrm{length}\mathrm{of}\mathrm{seedlings}\hfill \end{array}$$

Root colonization

Closed test tube assay was performed to access the root colonization ability of organism. In a sugar tube, sand was filled till 6 cm from the bottom and above that soil was filled till 4 cm and autoclaved. Seeds were surface sterilized (same as mentioned in water agar test) and were coated with BS 90 formulation. These seeds were sown in autoclaved sugar tube containing sand and soil and was sealed with parafilm. After 15 days, seedlings were uprooted and with the help of sterilized forceps, roots were excised and inoculated on nutrient agar plate to check colonization of bacteria. Plates were kept at 30 °C for 24 h [34].

Co-inoculation test

Co-inoculation testing was done to investigate if the marine Bacillus affected the growth of other native soil bacteria prior to its introduction into the soil. On nutrient agar, marine B. subtilis strain 90 had been streaked together with eight soil microorganisms that were isolated from chickpea field soil [35].

Pot trials

Talc-based formulation of BS 90 was prepared for coating the seeds. Seeds were first surface sterilized (same as mentioned in water agar test) and coated. Bioformulation was prepared by using talc, calcium carbonate, and carboxy methyl cellulose (CMC). 15 gm of calcium carbonate and 10 gm of CMC per kg of talc were mixed together and autoclaved in a sterile metal tray. In order to make the coating slurry, BS 90 (10⁶ cells/mL) and sterile talc were mixed. It was coated on the seeds, and 10 seeds were sown in each pot. Experimental setup is mentioned in Table 1.

Table 1.

Pot trial setup

Treatment 1	Control (No Coating)
Treatment 2	Coating with BS 90 bioformulation
Seed Variety	Hybrid Desi Chickpea
Replicates	3
Number of days trial conducted	28 days
Month of trial	June,2021
Average Day Temperature	32 °C
Average Night Temperature	27 °C
Average Humidity	78%

After 28 days, vegetative parameters were measured and effect on various stress markers was check.

Superoxide dismutase (SOD) estimation

The measurement of SOD activity was conducted using the procedure outlined by [36]. The presence of superoxide dismutase (SOD) hindered the reduction of nitro blue tetrazolium (NBT). Fresh root and leaves of treatment T1 and T2 were weighed (0.5 gm) and crushed in 2.5 mL of 0.1 M potassium phosphate buffer (pH 6) and centrifuged at 2000 X g for 10 min. Supernatant was used to determine SOD. To 0.1 mL of sample, equal volume (0.25 mL) of 50 µM riboflavin, 80 µM NBT, 12 mM methionine and 0.1 mM EDTA was added. To this mixture 2.15 mL of 0.1 M potassium phosphate buffer was added and the mixture was then exposed to sunlight for a duration of 20 min. After incubation, absorbance was measured at 560 nm using Shimadzu UV Visible-1800. Control tube (for calculation) was kept without adding sample. The percentage inhibition was calculated by following formula:

$$Percentageinhibtion=\frac{ControlOD-TestOD}{ControlOD}\times 100$$

Peroxidase (POX) Estimation

Sample preparation for POX estimation is same that of SOD. To 0.1 mL of supernatant, 2.4 mL of 0.1 M potassium phosphate buffer, 0.3 mL 5.33% pyrogallol and 0.20 mL hydrogen peroxide was added and mixed properly. In presence of POX, pyrogallol is converted to purpurogallin, is a brown coloured compound whose intensity is measured at 420 nm every 20 s for 1 min using Shimadzu UV Visible-1800 [37, 38]. POX activity is measured by the following formula:

$$POXactivity=\frac{\frac{\mathrm{\Delta }E420}{20sec}\times Finalvolume\times Dilutionfactor\times 1000}{Samplevolume\times 12}$$

where, 12 = absorbance of 1 mg/mL purpurogallin at 420 nm.

Proline estimation

Fresh root and leaves (0.5 gm) were crushed in 3% sulphosalycylic acid using mortar pestle and centrifuged at 2000 X g for 10 min. The supernatant was used for proline estimation. To 1 mL supernatant, equal volume of glacial acetic acid and acid ninhydrin (1.25 gm ninhydrin dissolved in 30 ml of glacial acetic acid and 20 ml of 6 N orthophosphoric acid) and kept in boiling water bath at 100 °C for 1 h. After the reaction is cooled down, 2 ml toluene was added and mixed for 1 min. Absorbance was measured at 520 nm using Shimadzu UV Visible-1800 [39]. Proline (Loba Chemie) was used as standard ranging from 10–100 µg/mL.

Phenol estimation

Fresh root and leaves (0.5 gm) were crushed in 10 ml methanol using mortar pestle and centrifuged at 2000 X g for 10 min. To 1 ml sample, 5 ml distilled water and 250 µL Folin–Ciocalteau reagent was added and incubated at 25 °C for 20 min and absorbance was measured at 725 nm using Shimadzu UV Visible-1800 [40]. Gallic acid was used as standard ranging from 10–100 µg/mL.

Pigment estimation

Fresh root and leaves (0.5 gm) were crushed in 10 ml acetone using mortar pestle and centrifuged at 2000 X g for 10 min. To 500 µL sample, 4.5 ml acetone was added and absorbance was measured at 470 nm, 663 nm, 646 nm using Shimadzu UV Visible-1800 [41]. Pigment was calculated using following formula:

$$\begin{array}{c}\mathrm{Chlorophyll}a=12.25A_{663}-279{\text{A}}_{646}\hfill \\ \mathrm{Chlorophyll}b=21.5A_{646}-5.1{\text{A}}_{663}\hfill \\ \text{Carotenoid}=\frac{1000A470-1.82Chlorophylla-85.02Chlorophyllb}{198}\hfill \end{array}$$

Statistical analysis

All the experiments were performed in triplicates. Pot trial was performed using randomized block design. All the results are represented as Mean ± SD. One way ANOVA and post havoc analysis were done using GraphPad Prism version 8.0.1.

Results

Bacterial strain 90 (BS 90) was isolated from marine water sample. BS 90 produced white coloured colonies and was able to grow on Marine Agar, Nutrient Agar, and Luria Bertani Agar. Results of biochemical tests are shown in Appendix 1. 16 s rDNA sequencing, NJ tree joining and phylogenetic analysis was done at Eurofins, Bangalore (Fig. 1).

Fig. 1.

Phylogenetic tree of BS 90 showing maximum similarity with Bacillus subtilis subsp. subtilis (NR 102783.2)

Sequence of BS 90 was submitted to GenBank nucleotide sequence database with accession number OP942238. BS 90 showed highest similarity with Bacillus subtilis subsp. subtilis strain 168 under accession number NR_102783.2 having bootstrap value 1000.

To optimise its growth, BS 90 was cultivated under different growth circumstances as shown in Fig. 2. BS 90 showed maximum growth in nutrient broth as compared to other media [Fig. 2 (a)]. Hence, further all tests were conducted using nutrient broth. Spectrophotometric analysis shows best growth at 0.5% salt concentration [Fig. 2 (b)], 8 pH [Fig. 2 (c)], 37˚ C [Fig. 2 (d)] and 150 rpm aeration [Fig. 2 (e)]. The isolate was found to be obligate aerobe, mesophilic.

Fig. 2.

Optimization of growth of BS 90 on different (a) media (b) salt concentration (c) pH (d) temperature and (e) aeration

The isolate was tested for several plant growth promoting traits which includes its ability to solubilize plant essential minerals, produce vital growth hormone like IAA and gibberellin and its ability to fix nitrogen. Qualitative analysis showed no IAA production but was found positive for gibberellin production. Quantitative analysis of gibberellin production showed 0.426 µg /mL production. Positive qualitative analysis for phosphate and potassium solubilization was found with solubilization indices of 1.30 ± 0.06 and 1.08 ± 0.011 respectively. A quantitative examination revealed the solubilization of 29.73 µg/mL of phosphate [Fig. 3 (a)] and 11.66 µg/mL of potassium [Fig. 3 (b)]. Additionally, nitrogen fixation was confirmed as positive. Figure 3(c) shows its growth on N-free Jensen media demonstrating that it could fix atmospheric nitrogen and use it to fuel its development. Peptone water was used to test the ammonia production. After three days, BS 90 produces ammonia at a rate of 0.84 mol/mL. BS 90 showed no HCN, siderophore production nor zinc solubilization.

Fig. 3.

a Phosphate solubilization (b) Potassium solubilization (c) Nitrogen fixation test

Gibberellins are crucial for promoting seed germination. Given the favourable test results for gibberellin production, the impact on seed germination was further investigated (Fig. 4). Seeds treated with BS 90 had 40% better germination than control seeds. Germination percentage and vigor index are shown in Table 2.

Fig. 4.

Chickpea germination after 7 days (a) Control (Untreated seeds) (b) Seeds treated with BS90

Table 2.

Effect of BS90 on germination and vigor index of chickpea seeds

Treatment	Germination %	Vigor Index
Control	60%	63.6
BS 90	100%	204

PGPR has a strong affinity for the rhizospheric region of the plant. Therefore, it happens to be simple for them to maintain communication with plant roots. The colonisation capacity of the marine isolate BS90 with roots was assessed. After 7 days of incubation, colonisation of BS 90 with chickpea roots was observed (Fig. 5). BS 90 showed colonization with chickpea roots after 24 h incubation.

Fig. 5.

a Chickpea seeds (triplicates) with BS90 treatment after 7 days of incubation (b) Colonization by BS90 with chickpea roots observed on nutrient agar plate

Since BS90 is isolated from marine water before using it as a biofertilizer in soil it is a necessary measurement to determine whether it is obstructing the growth of native soil bacteria. In the same nutrient agar plate, soil microorganisms isolated from chickpea field soil were streaked alongside BS90 for the dual culture test. The findings indicate that all soil microbes were able to develop remarkably well in the presence of BS90 and that neither the marine isolate BS90's own growth nor that of native soil microbes was impeded. As a result, despite being a marine isolation, it coexisted with other soil microorganisms to grow as shown in Fig. 6.

Fig. 6.

Dual culture plate assay showing growth of soil microbes (on sides) in presence of marine BS90 (centre)

Since BS 90 displayed a number of plant growth promoting characteristics, it was evaluated for growth promotion properties in vivo. A talc-based biofertilizer using BS 90 was developed and coated on chickpea seeds. The vegetative metrics (Table 3) along with several defence enzymes SOD, POX, Proline, Phenols, Chlorophyll and Carotenoid content (Fig. 7) with talc-based biofertilizer were substantially enhanced. Plant weight and height both increased by 14.17% and 13.71%, respectively, with the BS90 treatment. While root length, root hairs and leaves were observed to increase by 21.47%, 30.93% and 11.18% respectively. Hence, BS 90 formulation was able to assist in development of plant.

Table 3.

Vegetative parameters post pot trial after 28 days

	CONTROL	Bacillus subtilis
Total length (cm)	39.6 ± 0.754	45.03 ± 0.35***
Root length (cm)	3.40 ± 0.16	4.13 ± 0.25**
Shoot length (cm)	36.19 ± 0.66	40.9 ± 0.11***
No. of leaves	101.33 ± 2.51	112.66 ± 2.08***
No. of root hairs	32.333 ± 2.5	42.33 ± 2.5***
Total fresh mass (gm)	1.27 ± 0.03	1.45 ± 0.02***
Shoot fresh mass (gm)	1.124 ± 0.036	1.08 ± 0.06*
Root fresh mass (gm)	0.145 ± 0.005	0.37 ± 0.07**
Total dry mass (gm)	0.60 ± 0.065	0.77 ± 0.025*
Shoot dry mass (gm)	0.523 ± 0.066	0.68 ± 0.02*
Root dry mass (gm)	0.08 ± 0.002	0.093 ± 0.005*

^*p Value has been calculated using one-way ANOVA and its interpretation is as follows:

ns (p Value greater than 0.05) nonsignificant as compared to control

^* (p Value between 0.05 and 0.01) significant at 5% as compared to control

^** (p Value between 0.01 and 0.001) significant at 1% as compared to control

^*** (p Value less than 0.001) significant at 0.1% as compared to control

Fig. 7.

Effect of BS90 on (a) SOD, (b) POX, (c) Proline, (d) Phenol, (e) Chlorophyll-a, (f) Chlorophyll-b and (g) Carotenoid content of chickpea plant. *Note: p Value has been calculated using one-way ANOVA and its interpretation is as follows: ns (p Value greater than 0.05) nonsignificant as compared to control. * (p Value between 0.05 and 0.01) significant at 5%. ** (p Value between 0.01 and 0.001) significant at 1% as compared to control. *** (p Value less than 0.001) significant at 0.1% as compared to control

Pre and post sowing soil properties is shown in Table 4. Soil phosphate, potassium and nitrate content was observed to improve in BS 90 treatment. This may be because BS 90 is phosphate, potassium solubilizer and ammonia producer. Thus, while supporting plant growth it is also beneficial to soil.

Table 4.

Soil analysis (PRE AND POST POT TRIAL)

	PRE SOWING	POST SOWING
		CONTROL	BS 90
pH	8	8	8.5
Organic Carbon (%)	Medium (0.505–0.750)	Medium (0.505–0.750)	Medium (0.505–0.750)
Phosphate (kg/hectare)	Medium (22 to 56)	Medium (22 to 56)	Medium high (56–73)
Potassium (kg/hectare)	Medium low (Below 112)	Medium low (Below 112)	Medium (112–280)
Ammonical Nitrogen (kg/hectare)	Medium (About 73)	Medium (About 73)	Medium (About 73)
Nitrate (kg/hectare)	Low (About 10)	Low (About 10)	Medium (About 20)

Discussion

Microorganisms utilize the carbon and nutrients in organic matter to support their own growth as they metabolize it. Beneficial soil bacteria carry out essential tasks like breaking down crop wastes and nutrient cycling [42]. They discharge extra nutrients into the soil where plants can absorb them, promoting plant development. Such microorganisms are called plant growth promoter (PGP). PGPs are considered to regulate important growth-promoting pathways in the host plants, resulting in optimal growth maintenance, and have the ability to increase crop endurance to stressors [43, 44]. One of the most efficient methods for reducing biotic stress is the use of biocontrol agents as a viable, ecologically friendly option. Due to their capacity to colonize the rhizosphere and their capacity to actively combat the pathogen through a variety of mechanisms, antagonistic bacteria have been shown to be an effective technique for controlling soil-borne diseases [45, 46]. Particularly, as growth promoters, Bacillus spp. are acknowledged as one of the best and safe option [47].

In the present study, marine isolate BS 90 was confirmed to be Bacillus subtilis subsp. subtilis. BS90 could grow in stress conditions such as 8% salt, acidic pH 5, and temperatures as high as 42 °C. As a result, this organism may be used for crops growing in acidic soil, marine soil, and high-temperature regions. Additionally, it was discovered that BS90 was hemolytic negative and did not impair the growth of indigenous soil bacteria, which ensured its safe field application. A multi-trait plant growth promoting bacteria (PGPB) is an organism that demonstrates multiple features that promotes plant growth [48]. BS 90 was found to be phosphate and potassium solubilizer, ammonia and glucanase producer. The primary nutrients that are most scarce in the soil and that also have the highest beneficial effects on plants are nitrogen, phosphorus and potassium [49]. Nitrate is a vital component of plant growth and serves as a nutrient for plants. It aids in the synthesis of organic components like proteins and nucleic acids and is absorbed from the soil through the roots. Through their roots, plants take in nitrates from the water [50]. Fertilizers for plants contain significant amounts of nitrates. Nitrate serves as a signaling molecule and promotes seed germination and hence increases vigor index as well [51, 52]. BS 90 showed 40% increase in germination in chickpea seeds as compared to control and vigor index of 204. Also, the production of ammonia indirectly affects growth of plant as it is involved in the providing nitrogen to plants. Additionally, nitrogen fixation by BS90 has been found to be positive; as a result, it will be competent to raise the nitrogen content of plants. In addition to producing 0.84 µmol/mL of ammonia, BS 90 also raised the post-pot trial soil analysis's nitrate concentration. Thus, it can assist in giving plants nitrogen that they can use to promote their growth. Burkholderia and Stenotrophomonas sp. are also reported as nitrogen fixers that aid in the growth of plants [53]. Co-inoculation of Bacillus velezensis Strain S141 and Bradyrhizobium Strain tested as nitrogen fixers demonstrated an increase in plant weight by 22.9% in soyabean [54] while BS 90 solely exhibited a 14.17% rise in weight of chickpea. Furthermore, the availability of nutrients has a significant impact on chlorophyll concentration in leaves. The amount of chlorophyll declines in the absence of nitrates. The Calvin cycle proteins and thylakoids make up the majority of the nitrogen in leaves, which is why the photosynthetic capacity of leaves is connected to the nitrogen concentration. Thylakoid nitrogen is roughly proportionate to the amount of chlorophyll. The major pigment associated with the process of photosynthesis is chlorophyll a. Chlorophyll a content in the control plant was 6.25 mg/mL while it was 7.6 mg/mL in the BS 90-treated plant. The plant is anchored to the ground in a specific location via its roots. Whereas, root hairs are solitary tubular expansions of the plant root that increase the root surface area and improve water and nutrient uptake. Signals from root exudates can affect plant growth [55]. BS 90 formulation increases root height, weight and hair which directly helps in more uptake of nutrients which resulted in increase of biomass as well. It is also able to colonize with the root. This "anchorage" benefits the soil in addition to helping the plant perform other activities. A broad root system aids in keeping the soil in place so that wind and rain are less prone to damage it [56]. Along with photosynthetic pigment like chlorophyll, BS90 treatment also served in increasing carotenoids. Carotenoids play an important role in photosynthetic cells by serving as auxiliary light-harvesting pigments [57], extending the range of light absorption [58] along with having antioxidant activity [59, 60].

Most soils are deficient in accessible phosphorus content. One of the most important soil minerals for plants is phosphorus. It is a component of plant cells and is necessary for cell divisions and the growth of the plant's growing tip. It is essential for young plants and seedlings [61]. Phosphorus enhances tillering, encourages early root development, and seed development, and improves water usage efficiency. In plant tissue, potassium is involved in the flow of water, minerals, and carbohydrates. It has a role in the plant's enzyme activation, which has an impact on the synthesis of protein, starch, and adenosine triphosphate (ATP). The process of photosynthesis can be controlled by ATP synthesis [7]. The transport and distribution of products from photosynthetic processes are significantly impacted by the potassium status of plants [62]. Such macronutrients as phosphate and potassium are solubilized by BS 90 and converted into soluble forms that are easier for plants to absorb. Gluconacetobacter diazotrophicus PAl 5 is reported to solubilize 12.67 µg/mL of phosphate [53] whereas Bacillus strain IA7 has potential to solubilize 2.75 μg/mL phosphate [63]. It was observed during the lab experiment that BS 90 solubilizes 29.73 µg/mL of phosphate and 11.66 µg/mL of potassium. After sowing seeds with BS 90 bioformulation, it also raised the levels of phosphate and potassium in the soil. Hence, this organism has the ability to provide optimal nutrients to plant from soil. BS 90 was also found to colonize root which enhances the host plant's capability to absorb mineral nutrients, particularly phosphorus and nitrogen.

Defense marker like SOD and pigment carotenoid protects plant from oxidative stress. Oxidative stress is a result of an imbalance between the generation of reactive oxygen species (ROS) and their removal [64]. It causes photooxidative deterioration of DNA, proteins, and lipids as a result of more net ROS generation, that eventually leads to cellular death [65]. BS90 treatment showed increase in SOD and carotenoids which assist in removal of ROS hence, protecting plant from oxidative stress [66, 67]. SOD, in addition to serving in the primary defence mechanism in plants, also exerts influence on plant growth and development. SOD plays a crucial role in controlling the process of flowering by regulating the equilibrium of reactive oxygen species (ROS) in plants [68], maintaining normal plant growth and improving stress tolerance [69]. Along with these, increase in POX and total phenol content was also observed. POX and phenol increase lignin synthesis in plants [70]. The building blocks of lignin, called monolignols, are formed in the cytosol, transferred to cell walls, and then polymerized through oxidative processes mediated by POX [71] whereas phenolic compounds function as a source of phenylpropanoid units for the production of lignin [72]. Lignin is one of the major components of plant cell wall providing strength to the plant cell. Proline accumulation in non-stress conditions as observed in BS90 treated plants is also involved in cell wall synthesis as it is a vital amino acid in many cell wall proteins [73]. Proline is an amino acid that is produced by plants in response to both stressful and non-stressful environments. It serves as a helpful solute for the plants. Proline serves a key role in the development and differentiation of plants throughout their life cycle. It is a crucial factor that influences the characteristics of several cell wall proteins and performs significant functions in the growth and development of plants. Extensins, arabinogalactan proteins, and hydroxyproline- and proline-rich proteins are crucial constituents of cell wall proteins. They have significant functions in cell wall signal transduction cascades, plant growth [73], and safeguarding cells from oxidative stress [74].

Conclusion

In the current work, a marine isolate was isolated from Gulf of Khambhat, Gujarat, India. This marine isolate was identified as Bacillus subtilis strain BS 90 and it was found to stimulate chickpea development, suggesting that it might be used as a plant growth promoter. Furthermore, it was found that BS 90 treatment resulted in an induction of biomass during pot trials along with positive effect on pigments and stress markers. The potential of marine bacteria to boost plant growth has been demonstrated, but more investigation is necessary to fully explore the molecular connections as well as its mode of action, in order to develop an efficient fertilizer.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

K. Rathod is thankful to CHARUSAT and SHODH, Government of Gujarat for providing doctorate fellowship.

Data Availability

The data will be available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare that they have no competing interests.