Impact of Lysinibacillus macroides , a potential plant growth promoting rhizobacteria on growth, yield and nutritional value of tomato plant ( Solanum lycopersicum L. F1 hybrid Sachriya)

Plant growth promoting bacteria enhance the growth in plants by solubilizing insoluble minerals, producing phytohormones and by secreting enzymes that resist pathogen attack. The present study was aimed at identifying the potential of Lysinibacillus macroides isolated from pea plant possessing rich microbial rhizobiome diversity in promoting the growth of tomato plant ( Solanum lycopersicum L.). Potential of L. macroides in the promotion of S. lycopersicum L. growth by increased shoot length, terminal leaf length and breadth was assessed. Anatomical sectioning of stem and root revealed no varied cellular pattern indicating that the supplemented bioculture is not toxic to S. lycopersicum . Plantlets treated with L. macroides along with organic compost showed an increased total phenol content (17.58±0.4 mg/gm) compared to control samples (12.44±0.41 mg/g). Carbohydrate content was noticed to be around 1.3 folds higher in the L. macroides plus compost mixture supplemented slots compared to control sample. Significant increase in shoot length was evident in the L. macroides plus compost supplied slots (23.4±2.7 cm). Plant growth promoting properties might be due to the nitrogen fixing activity of the bacteria which enrich the soil composition along with the nutrients supplied by the organic compost. Rich microbial rhizobiome diversity in pea plant and the usage of L. macroides from a non-conventional source improves the diversity of the available PGPR for agricultural practices. Further research is needed to detect the mechanism of growth promotion and to explore the plant microbe interaction pathway.


Introduction
With the expectancy of the global population rising to 9 billion by 2040, there is a major threat faced by the developing countries to address the problems associated with crop production (1). Meeting the demand for high yielding crop varieties with enhanced nutritional content is the need of the hr for agriculture agencies. Better quality and improved productivity of crop plants is possible by replacing the lost nutrients in the cultivable land (2). Solanum lycopersicum, one of the most cultivated fruits worldwide for its nutrients such as lycopene, βcarotene, flavonoids and vitamin C are known to have high market value for their antioxidant properties (3). It is used in various cuisines of the world and is also used as a model plant for fleshy fruited dicots (4).
Farming practices using chemical fertilizers for long tenure decreases the soil fertility and agricultural productivity thereby affecting the natural process of nitrogen fixation in the soil (5,6). As an alternative, organic fertilizers and biofertilizers provide an ecofriendly approach by improving nutrient uptake, water absorption, water retainability, atmospheric nitrogen fixation, phosphate solubilisation (7,8). Plant benefiting microorganisms grouped as Plant Growth Promoting Rhizobacteria (PGPR) offer a wide range of benefits by colonising the root and promote plant growth by improving the accessibility to bionutrients, solubilisation of inorganic phosphate and by limiting plant pathogens (3). PGPR confers growth benefits to plants either directly or indirectly (9). PGPR act directly by aiding nitrogen fixation, enhancing iron uptake, secreting organic acids, solubilizing insoluble phosphate and by producing phytohormones (10)(11)(12). Indirect mode of action is by producing protease kind of enzymes that resist fungal attack and chemicals like hydrogen cyanide (HCN) that inhibit pathogen growth (12,13,14). Several diverse genera of microbes are employed as PGPR including Bacillus, Pseudomonas, Arthrobacter, Paenibacillus and Lysinibacillus (15,16). Bacillus cereus and Klebsiella variicola isolated from the rhizospheric region of tomato capable of secreting phytohormones such as GA3, IAA and kinetin enhanced mineral uptake and upregulated chlorophyll synthesis (17). Lysinibacillus reported for its ability to tolerate metal has been tested for its efficacy to promote plant growth promotion in polluted environment (18). L. macrolides, supplemented individually or as a consortium culture conferred antimicrobial activity against plant pathogen Xanthomonas campestris, causative bacterium that is known to cause black rot of cabbage (19). Total nitrogen content in the soil can be enhanced by L. macroides by its ability to fix atmospheric nitrogen and decompose organic matter (20). Nitrfying bacteria convert the atmospheric nitrogen (N2) into organic nitrogen which can be utilized by plants. Certain bacteria like Providencia spp. enhance plant growth by secreting phytohormones such as indole acetic acid (21,22). Microbe and its secretory products are widely employed in disease inhibition and shelf life extension. Artificial introduction of microbial antagonists found to be more efficient in disease control and exhibited limited pathogen attack (23,24). A combination of technological advances in agricultural farming comprising both biofertilizers and postharvest treatment will ensure better yield in agriculture (25). Unexplored microbial community from the pea plant cultivated in the agricultural ecosystem result in identification of novel diverse rhizosphere microbiomes for agricultural practices. Our study is focused on identifying a novel plant growth promoting bacteria from the rhizosphere of pea plant and their efficacy in enhancing the growth of S. lycopersicum L. was evaluated.

Isolation and Identification of PGPR from Pisum sativum
Rhizospheric soil sample was collected from P. sativum propagated in an agricultural ecosystem near IIHR (Indian Institute of Horticultural Sciences), Bengaluru, India. The sample (1 gm) was suspended in 10 ml of sterile water and was serially diluted. The diluted sample was spread plated onto yeast extract mannitol agar (YEMA) and incubated for 24 hrs at 37°C (22). The isolates obtained were screened for its nitrogen fixing, phosphate and zinc solubilizing ability (16,26,27). Culture was biochemically characterised using Gram's staining, IMViC tests and screened for its ability to produce ammonia, hydrogen cyanide and siderophores (28,29). The 16S rDNA gene from the isolated genomic DNA was amplified using 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3' primers. The resulting amplicon was purified and sequenced using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer and the obtained sequence was compared with the homology search tool BLAST in NCBI genbank database for species identification. A phylogenetic tree was constructed using Mega-X version 10.2.4 using the maximum likelihood method based on the Kimura 2-parameter model (30)(31)(32).

Experimental design
A total of five treatment groups along with a control were designed for the application study (Table. 1). Pseudomonas fluorescens procured from the culture collection center, GKVK Bangalore, India served as a positive control. P. fluorescens is a PGPR well known for its ability to fix atmospheric nitrogen, production of plant growth promoting compounds (IAA) and production of antimicrobial agents such as bacteriocins, antibiotics, and siderophores. Compost was prepared by packing multiple layers of food and vegetable waste over a layer of crushed dried leaves in a compost pit dug to one feet depth. The leaves and waste were laid in 1:1 ratio and the pit was covered with soil and charcoal to facilitate composting. After 30 days, the contents from the pit were mixed with dry cow dung and were utilized as compost.

Seed germination
Seeds of tomato plant (Solanum lycopersicum F1 hybrid Sachriya) were incubated for 1 hr in a broth culture of L. macroides (8 Log CFU/ml) and P. fluorescens (8 Log CFU/ml) separately. Treated and control seeds were planted in a coco peat for germination. After 7 days, plantlets were transferred to pots (33). Inoculation of bioculture was initiated based on the concentration mentioned in Table 1. The study was conducted for a period of thirty days. The bioculture (1ml) was supplemented to the plantlets at every two days interval and was maintained in a controlled environment in a polyhouse.

Growth and anatomy assessment
The effects of the bioculture treatment on plant growth was analyzed for a period of 4 weeks by measuring the shoot length, leaf number, terminal leaf length and breadth every week (34). To check the effect of the PGPR on the vascular bundles and to determine whether the PGPR invades the root and stem, anatomical variations in control and the treatment group was carried out by analysing the transverse section of stem and root. Thin sections in 366 JYOLSNA ET AL.

Total chlorophyll estimation
Leaf sample (500 mg) obtained from each treatment group was homogenised using 10 ml of 80% acetone and centrifuged at 10000 rpm for 10 min. at 4 °C. The supernatant was collected and the absorbance was measured at 645 nm and 663 nm and chlorophyll content was estimated (35).

Protein estimation
Protein content was estimated using the Bradford assay (36). Leaf samples (500 mg) from each treatment group were homogenised in 10 ml (0.05 M) phosphate buffer and centrifuged at 10000 rpm for 10 min. at 4 ˚C. 0.1 ml of supernatant was made up to 1ml using distilled water and 5 ml of Bradford reagent was added to the test tubes including the blank (1ml of d.H2O) and the absorbance was measured at 595 nm. A standard calibration curve was prepared using Bovine serum albumin (BSA).

Carbohydrate estimation
Carbohydrate was estimated using phenol-sulphuric acid method (37). Leaf sample (100 mg) was homogenised with 5 ml of 2.5N HCl and was kept in a boiling water bath for duration of 3 hrs. After cooling, sample was neutralized by adding solid sodium carbonate powder till the effervescence ceased and centrifuged at 10000 rpm for 10 min. 200 μl of the supernatant was made up to 1 ml with distilled water. 5 ml of 96% sulphuric acid and 2% phenol were added in a cold water bath and the absorbance was measured at 490 nm. A standard calibration curve was prepared using D-glucose.

Determination of Antioxidant activity
Antioxidant activity was estimated using DPPH scavenging assay (38). 0.1 gm of powdered leaf samples were weighed and ground with 2 ml methanol using mortar and pestle. The solution was transferred to eppendorf tubes and centrifuged at 10000 rpm for 20 min. 20 μl (0.46 mg) of the extract was made up to 3 ml with methanol. 1 ml of DPPH [0.004% (w/v)] was added and incubated for 30 min. in dark condition. Absorbance was measured at 513 nm and the antioxidant activity was calculated (38).

Total phenolic content and tannin estimation (TPC)
The total phenolic content (TPC) and tannin estimation was performed using the Folin-Ciocalteu assay (39). For TPC, 0.1 gm of the powdered leaf sample was weighed and ground with 2 ml of methanol in mortar and pestle. 50 μl of the supernatant was made upto 3 ml with distilled water. To the mixture, 0.5 ml of the Folin-Ciocalteu reagent (1:1) was added followed by mixing with 2 ml of 20% sodium carbonate solution. Absorbance was measured at 638 nm after 3-5 min. incubation in a dark environment. Similarly for tannin estimation, 0.1 gm of the powdered leaf sample was weighed and ground with 2 ml of methanol in mortar and pestle. 50 μl of the supernatant was made upto 1 ml with distilled water. 0.5 ml of the Folin-Ciocalteu reagent (1:9) was added followed by addition of 2 ml of 20 % sodium carbonate solution and the absorbance was recorded at 725 nm after 40 min. incubation in dark. The calibration curves for TPC and tannin were prepared using catechol and tannic acid respectively.

Flavonoid estimation
Flavonoid content was measured by modified aluminium chloride colorimetric method (40). 0.1 gm of the powdered leaf sample was weighed and ground with 2 ml of methanol in mortar and pestle. 50 μl of the supernatant was mixed with 0.1 ml of 10% aluminium chloride and 0.1 ml of 1M potassium acetate. Absorbance at 415 nm was recorded after 40 min. incubation. A standard calibration curve was prepared using quercetin.
Note: Drying of leaves was carried out carefully without exposing the samples to direct sunlight in order to prevent loss of secondary metabolites. Absorbance for all values was measured using the Shimadzu UV-160A spectrophotometer.

Statistical analysis
All statistical analysis were carried out using online freeware ASTATSA and advanced tool pak VBA. One way Analysis of variance (ANOVA) was performed with Tukey HSD, Scheffé, Bonferroni and Holm comparisons and a p-value <0.05 was considered significant.  (42). Results of this study analysed the effect of L. macroides infused compost made of vegetable and food waste on growth and yield of S. lycopersicum.

Isolation, screening and identification of PGPR
The isolate obtained was biochemically characterised (Table. 2) and was identified as L. macroides (GENBANK accession number; MK517553) after performing a BLAST search with a maximum score of 1967 on a 99.36% query cover. A molecular phylogenetic tree was constructed using MEGA-X Version 10.2.4 by maximum likelihood method based on Kimura 2 parameter (Fig. 1).

Growth Assessment and anatomical examination of S. lycopersicum
Growth enhancement in plants is attributed to good soil conditions that can be further enhanced by the application of organic, biofertilizers and PGPR (46). Compost supplemented with biofertilizer acts as an efficient soil amendment factor that facilitates water and mineral (nitrogen and phosphorus) uptake. Beneficial plant nutrients obtained from compost blended with nitrogen fixing bacteria and improve the availability of nitrogen sources for amino acid synthesis (47). In this study, growth measurements of Solanum lycopersicum was assessed for a span of thirty days and the study identified maximum shoot length and terminal leaf length in groups supplemented with L. macroides (p<0.05) (Fig. 2, Fig.  3 respectively). Increased stomatal conductance in PGPR supplied plants confers better water-use efficiency in PGPR supplemented plants (48). Terminal leaf breadth was highest in the group supplemented with compost only (p<0.05) (Fig. 4). Growth enhancement in compost and L. macroides supplemented slots can be attributed for better nutrient enrichment in soil conditions. The effect of PGPR Pseudomonas fluorescens supplemented along with organic manure containing composted material of vegetable and fruit sources enhanced the growth and yield of maize plant (46). Treatment of tomato plant with phytohormone secreting Sphingomonas sp exhibited remarkable growth and physiological characteristics such as stimulated plant cell division, enhanced root formation and shoot elongation (49). In our research, maximum number of leaves was observed in plant groups supplemented with compost plus L. macroides by the end of 21 days (Fig. 5). This enhancement could be due to the upregulation of molecules    secreted by the bioculture that mediate phytostimulation. The transverse section of stem and root of control and the most effective treatment group (compost supplemented with L. macroides) was assessed. PGPR supplementation to S. lycopersicum increases the number and diameter of cortical cells and vascular tissue which could be attributed to the enhanced nutrient and mineral uptake (50,51). Nutrient deficiency and stress leads to reduced size of vascular bundles affecting the overall plant health (52,53). An increase in pith diameter was noted in the treatment groups whereas size of the cortical cells and xylem vessels remain the same in all groups. Pathogenic effects such as abnormal vasculature or any visual deformities because of PGPR supplementation affecting the morphology and anatomy of S. lycopersicum was not observed throughout the treatment period (Fig. 6).

Estimation of biochemical constituents and phytochemicals
Efficacy of L. macroides on the synthesis of plant pigment chlorophyll, carbohydrate and protein were assessed upon treatment with biofertilizer and compost (Table. 3). Maximum biochemical constituents were observed in compost and biofertilizer supplemented samples (p<0.05). Specific rates of nitrogen supply increase the accumulation of photosynthetic pigments. Higher pigment synthesis could be due to enhanced nitrogen fixation caused by the inoculated PGPR. Nitrogen constitutes the major component of the chlorophyll structure. Bacillus pumilus enhances the synthesis of soluble proteins in soybean plants (54). Combined or sole application of organic manures and biofertilizers increases the yield of the phenolic compounds and phyto components that influence the quality attributes in the tested plant samples. Appropriate usage of L. macroides along with compost material increased solubility and bioabsorbtion of nutrients in the current study. Similar results were obtained in Brassica oleracea treated with P. fluorescens and humic acid combinations (55,56). Co-inoculation of plant growth promoting rhizobacteria along with farm yard compost enhances the bioaccumulation of nutrients in the mungbean by aiding in nutrient fixation, release, uptake and transportation (57).
Phenolic compounds synthesised by plants contribute for growth and development and involve in various metabolic activities such as plant protection from free radical attack, protection against plant invading pathogens and as signalling messengers (58). In this study, secondary metabolites like polyphenols, flavonoids and tannins were estimated in control and L. macroides supplemented plant samples (Table. 4). Supply of L. macroides alone and L. macroides co-inoculated with compost slot exhibited higher synthesis of flavonoid. No significant differences on flavonoid synthesis were noted between the Pseudomonas and Pseudomonas plus compost supplemented groups. Difference in the synthesis of flavonoid between the treatment groups could be due to the factor associated with the nutrient breakdown and uptake ability of PGPR amended with the compost. Enhanced phenolic content in Serratia marcescens supplemented Piper belte L. was noted (59). Parallel results were observed in chickpea by inoculation of PGPR P. fluorescens and P. aeruginosa (60). Enhanced flavonoid concentration was observed in compost and Lysinibacillus macroides co-inoculated compost samples. PGPR stimulates the phenylpropanoid pathway for enhanced polyphenols synthesis thereby contributing to host defense against plant pathogens (61). Induction of polyphenols by compost supplementation along with bacterization using L. macroides increased the total phenolic content in the S. lycopersicum. Results of the study suggest that the use of organic fertilizers along with PGPR can enhance the production of plant secondary metabolites. Tannin accumulation is one the primary ways the plant system adopts to avoid stress induced changes. Higher synthesis of tannins and other phenolics are reported for radical quenching activity in Acacia gerrardii supplemented with Bacillus  subtilis and Mycorrhizal fungi (62). The aromatic oxygen substituted derivatives such as tannins and flavonoids contribute for homeostasis and health maintenance (63). PGPRs influence plant growth, nutrient utilization by producing metabolites such as flavonoids, phenols, saponins and alkaloids that benefit plant growth and stimulation (24).

Antioxidant activity
Oxidative stress can reduce the nutritional quality of crops. Environmental factors such as air pollution, extensive herbicide/pesticide application, contamination by heavy metal, drought, salinity, injuries can induce oxidative stress in plants. Use of the PGPR can reduce the negative effects caused due to environmental stress by enhancing the antioxidant state (57). PGPR application aids in plant growth by reducing the effect of cold stress and also by activating the enzymes that reduce Reactive Oxygen Species (ROS) (58). In our study, maximum antioxidant activity was observed in plants supplemented with P. fluorescens (76.55±1.45%) compared to other treatments by 4 th week (Fig. 7). Enhanced DPPH radical quenching activity was noted in S. lycopersicum samples treated with compost infused with plant growth promoting bacteria. Mechanism related to enhanced antioxidant activity could be due to the elevation in the expression of antioxidant genes that confer homeostasis in the plant metabolism (64,65). Cucumis sativus infused with Promicro monospora, Burkholdera cepacia, and Acinetobacter calcoaceticus showed enhanced phenol production due to upregulation of antioxidant enzymes (66).

Conclusion
Development of a cost effective, eco-friendly approach for enhancing plant growth is a much needed practice towards developing a productive agricultural ecosystem. Identification and development of functional soil amendments will minimise the usage of chemical fertilisers thereby nourishing the soil with less artificial pollutants. In our study, supplementation of Lysinibacillus macroides infused compost exhibited a promising effect in enhancing the overall plant growth. However, molecular mechanisms that govern the interaction of PGPR with host biomolecules need to be further characterized. Identification and utilisation of novel PGPRs with potential applications in agriculture will increase crop productivity and minimize negative impacts of organic waste.  Phosphate solubilization Negative 10.
Nitrogen fixation Positive  Note:: All the parameters are significant at p<0.05 and indicated by *.