Physiological and Biochemical Attributes of an Endophyte Stenotrophomonas maltophila, AVSW 1 Isolated from Chilli on   PGP of Tomato

Introduction

Tomato (Lycopersicon  esculentum Mill) is one of the rich source of vitamins, minerals, organic acids, essential amino acids and dietary fibres.Abiotic and Biotic factors(microbes) decreasing the production of tomato crop. Many Microbial diseases like Fusarium wilt, Early blight, Late blight, damping off, Leaf spots, Bacterial fruit canker, Anthracnose, bacterial speck affecting Tomato crop. Previous studies reports that ,soil nitrogen deficiency is decreasing quality and production of tomato crop, which can be reclaimed by potential PGP Bacteria as an alternative and eco friendly alternative to chemical fertilizers ^1-5. Nowadays, bio inoculants have gained more global attention due to their eco-friendly, cost-effective and easily replicable nature and used in agricultural ecosystems because of the presence of plethora of secondary metabolites responsible for plant growth promotion ,stress tolerance and disease resistance ^6-11. Endophytic bacteria enhances plant growth by the production of IAA, Ammonia, Siderophore, secondary metabolites, solubilizing Phosphate, increasing pathogenicity and maintain the integrity of host cell wall^1,2,12,13. Due to  its close association with the host, they developed a unique mechanism of synthesizing secondary metabolites for amicable association with the host in a symbiotic manner. Such specific secondary metabolites and volatile compounds express defence against fungi and bacteria ,induce systemic resistance, immunity and enhance plant growth under oxidative stress conditions^14-19.

Siderophores produced  by plant growth promoting bacteria are also involved in iron transportation and as biocontrol agents by scavenging the available iron present in the surroundings of the pathogen siderophore production ²⁰. However, the metabolic profiling and physiological attributes of metabolites  from  endophytic bacteria have not been much focussed. The present research aimed to understand the metabolic profiling of an endophytic bacteria, Stenotrophomonas maltophilia isolate, extracted from green fruits of chilli and its physiological attributes in the growth promotion of tomato.

Isolation of Stenotrophomonas maltophila AVSW 1 

AVSW 1 an endophytic bacteria isolated from the Green fruit of the chilli plant, was collected from Guntur, Andhra Pradesh, India . It was collected  from the corresponding author as a subject for the current research.AVSW 1 was purified by the streak plate method and grown in nutrient broth. After 48 hrs of incubation, the endophytic bacterial strain was stored ^21-23.

Materials and Methods

Screening of Fungal Antagonism and PGP Activities 

The antifungal activity of AVSW 1 was assessed using the double culture method ²⁴. Bacterial isolates were streaked on Sabouraud dextrose sugar (SDA) medium at 3 cm from the pathogenic fungi inoculated at the centre. Antifungal activity was measured at room temperature  after 4-7 days of incubation. Inhibition levels were measured as I= 1-(a/b) X as described earlier^{13-17, 25,26.}  

Indole-3-acetic Acid Production

One ml culture filtrate of 72 hrs old bacterial culture was supplemented with 1 μg. ml^{– l} L- tryptophan and 2 ml Salkowski reagent and incubated at 28 ± 20° C for 30 min. Appearance of Pink colour indicates the presence of IAA^27.

Phosphate Solubilization

Solubilization of Tricalcium phosphate was detected in Pikovskaya’s agar ²¹. Bacterial isolate was streaked on the surface of the Pikovskaya agar medium, and activity was estimated after 1 to 5 days of incubation at room temperature. Development of the clear zone around the bacterial colony was a positive response for phosphate solubilization.

Ammonia Production

Fresh  bacterial culture was inoculated  into 10 ml of peptone water and incubated at 36 ± 2° C for 48-72 h.A change of colour from brown to yellow with the addition of  0.5 ml of  Nessler’s reagent was a positive reaction for ammonia production ²⁸_.

Siderophore Production

The bacterial strain was grown on a succinate medium and incubated for 24-30 h with constant shaking at 120 rpm at 28⁰ C. For every 20 minutes of interval, 5 ml broth was taken and centrifuged at 10,000 rpm for 10 min at 4⁰ C. The cell-free supernatant was mixed with 0.5 ml CAS solution and colour was measured at 630 nm.0.5 ml un- inoculated succinate medium and 0.5 ml CAS solution was used as control ²⁸.

Biochemical  Physiological and Molecular Identification

Biochemical tests such as The IMViC test, Indole test, MRVP, Citrate utilization, hydrogen sulphide production, and sugar fermentation were done as per the standard protocols ²⁹.

Physiological Characteristics

Amylase,Cellulase, Catalase, Oxidase,Urease, Gelatin hydrolysis, Nitrate reduction, Haemolysis and Lecithinase  were analysed as per the protocols ^29-32.

Molecular Identification 

It was done according to Bergey’s manual of determinative bacteriology ³³ for tentative identification of Genus followed by 16S rRNA  partial gene sequencing analysis using universal primer 1492R (5´ TACGGYTACCTTGTTACGACTT-3′) and 27F (5′ AGAGTTTGATCMTGGCTCAG-3′). Phylogenetic tree analysis was done by using Neighbour joining method with 1000 bootstrap replicates. Isolate was deposited in GenBank, NCBI ³⁴.

Optimization Studies  

Physical parameters ,temperature, pH, incubation period, and chemical parameters  NaCl, Carbon ,and nitrogen sources were studied in 10ml of broth inoculated with100 μl of bacterial culture and incubated for 48 h .O.D of the culture broth was read at 600 nm ³⁵. Growth optimization of endophytic bacteria was analyzed at different temperatures ranging from 20-45°C at an interval of 5°C ,different pH value range [3-9] ,saline concentration range 1-6 % , 1% carbon sources ( Glucose, Fructose, Sucrose, Lactose, Maltose, Glycerol, Mannitol, Starch, and Cellulose), 0,5%nitrogen sources (KNO3, NaNO3, NH4SO2, Urea, Peptone, Beef extract, Yeast extract, Casein, and Malt extract) and  incubation periods (24 h, 36 h, 48 h, 60 h and 72 h).Bacterial Growth Curve was also determined for 24 h bacterial culture at an interval of 4 h .

PGP Activity of Endophyte by Pot Assay Studies: 

Tomato seeds were treated with 48 h bacterial culture for 30 min and shade-dried for 1 h. Inoculated seeds were seeded into coco peat,and the pots were kept under greenhouse conditions.% of seed germination was evaluated at an interval of 4,6 and 8 weeks. Physiological parameters, root length, shoot height ,biomass, no of leaves, no of lateral roots, and chemical constituents Protein content by Lowry’s and carbohydrate  content  by DNS method were estimated.

Metabolite profile Finger Print of Endophytic Bacteria

Secondary metabolite finger print of endophyte was analysed in ethyl acetate extract using Fourier transforms infrared spectroscopy (FTIR) and GC-MS^36,37,39

Scanning Electron Microscopy

Root colonization ability was studied using SEM. One cm of root pieces were fixed and then processed with the PATOTO method. The prepared samples were mounted on aluminium stubs with Scotch TM double-sided tape, coated with gold in a sputtering Hummer II (Technics,Springfield,VA) and examined in a Cambridge S360 Scanning Electron Microscope.

Results and Discussion   

Fungal Antagonism and PGP Traits 

Pure culture of AVSW 1 was screened for fungal antagonism against Fusarium oxysporum  by dual culture method as shown in Plate -1. AVSW 1 showed significant inhibition of  Fusarium oxysporum compared to control and positive to PGP traits such as IAA, Ammonia, Phosphate solubilisation and siderophore production in qualitative screening.

Plate 1: Screening of Fungal antagonism of AVSW 1 against Fusarium oxysporum. Click here to view Plate

Effect of Physical and Chemical Parameters on Bacterial Growth:

Physicochemical parameters were analyzed on a one-time factor (OFOT) basis to optimize the growth and maximise the production of PGP traits. Optimization studies revealed that AVSW 1 showed maximum growth at 37 ⁰ C, 1% salinity and 60 h incubation period with minimal medium ameliorated with 1% Fructose and 1 %Peptone (Fig 1).

The growth curve of S. maltophilia AVSW 1 showed 4-8 h of Lag phase,12-44 h of Exponential phase, and 44-68 h of  stationary phase. After 68 h, the decline phase was observed (Fig2).

Figure 1: Optimization of Physical and Chemical parameters on  growth of AVSW 1. Click here to view Figure

Figure 2: Growth analysis of AVSW 1 from 4 – 96 h of incubation in optimized medium. Click here to view Figure

PGP Traits of AVSW 1 

Plant growth promotion (PGP) traits in  S. maltophilia AVSW 1, such as IAA, Ammonia, PO4- solubilization and siderophore production, were evaluated after optimization.(Table1 and fig2 ) In S. maltophilia AVSW 1,IAA production increased by 55.66 % ,Ammonia production increased by 74.85 % ,Phosphate (po4^– ) solubilization  increased by 69.42  % and Siderophore production increased by 49.62 % after Optimization. Whereas in earlier findings, S. maltophilia JVB5, root endophyte of sunflower showed optimization were IAA (30.0 μg/ml) ,Phosphate solubilisation (32.23 ppm) and Siderophore (79.90 psu ) after optimization ⁴. Previous findings reported that two S.maltophila  IPR-Pv696 and 262XG2 root nodular endophyte strains of clover reported  IAA (30.26 μg /ml  and 31.15 μg/ml), Phosphate solubilization (100 and 75%) and phosphate liberated (278 mg/l and 208 mg/l )¹¹.10.73 μg /ml phosphate solubilization, Indole acetic acid 3.16 μg/ml and gibberellic Acid 40 μg /ml were observed in S. maltophilia , SBP-9 rhizobacteria of Sorghum bicolour at pH 8, temperature 30 ⁰ C,Nacl Concentration 6 % and broth incubated for 48 h ⁶. 818 ppm Phosphate solubilisation, 1,62 IU /ml Acid phosphatase , 93µg/ml IAA production, Ammonia production (80 µg/ml) and ability to produce  siderophore  and HCN reported earlier in Stenotrophomonas maltophilia AVP 27 rhizobacterium of chilli ⁴⁰. Similarly, present results also revealed that S.maltophila AVSW 1 is specific to the host in the expression of PGP traits and varies with endophytic and rhizospheric habitats. Further observed that the ability of phosphate solubilisation is less in endophytic strain compared to rhizosphere strain. It may be a marker characteristic feature to distinguish endophytic PGP strain from PGPR.

Table 1: Quantitative analysis of Plant growth promotion (PGP) traits in AVSW 1.

Optimization	IAA (μg/ml)	Ammonia (μg/ml)	PO4– solubilization (ppm)	Siderophore (psu)
Before (control)	14.17	15.07	33.6	33.33
After	25.1	33.1	69.33	55.33

Figure 3: Impact of optimization on Plant growth promotion (PGP) traits of AVSW 1  Click here to view Figure

Identification of AVSW 1     

Based on colony morphology, morphological, and physiological characteristics (Table 2) followed by 16s rRNA partial gene sequencing analysis (Fig 4), AVSW 1 was identified as Stenotrophomonas maltophilia and deposited in NCBI GENBANK with the name Stenotrophomonas maltophilia AVSW 1 with the accession no OQ293900 

Table 2:  Colony morphology and Morphological, Biochemical and physiological Characteristics of  AVSW 1     

Morphological Characteristics
Colony Morphology	Small, circular and off white
Gram’s reaction	Positive
Cell shape	Rod
Biochemical Characteristics
Indole ,MR	Negative
VP/Citrate	positive
Sugar fermentations
Glucose /Sucrose/Fructose/Maltose/Mannitol	A+/G–
Lactose / Arabinose / Inositol / Sorbitol/Dulcitol	A–/G–
Physiological chaacteristics
Decarboxylation reactions
Ornithine/ Arginine / Creatinine/ Lysine	Positive
Malonate	Negative
Enzymatic reactions
Amylase/Urease /Super oxide dismutase/ Phenylalanine deaminase /Gelatin hydrolysis/ Oxidase	Negative
Cellulase peroxidases Poly phenyl oxidase Lecithinase Catalase Oxidase Nitrate reduction	positive
Haemolysis	Positive
Spore formation	Positive

Figure 4: Phylogenetic distance tree of Stenotrophomonas  maltophilia  AVSW  1 ( OQ293900) constructed by the neighbour-joining method using Blast N of NCBI  with 1000 bootstraps  Click here to view Figure

Greenhouse Studies   

The plant growth promotion potential of Stenotrophomonas maltophilia AVSW 1 was tested on tomato seedlings using the seed bacterization method followed by inoculation of culture at the collar region under greenhouse conditions. Root colonization efficacy of AVSW 1 inoculum was observed on 4, 6 and 8week old seedlings treated with AVSW 1 under scanning electron microscope (Fig 5.A & 5.B).

Figure 5A: Scanning Electron Microgram of the root of tomato seedling treated with  AVSW 1. Click here to view Figure

Figure 5B: Scanning  Electron  Microgram showing clear root Colonization in AVSW 1 treated tomato seedlings        Click here to view Figure

Growth parameters such as root length,  shoot height, root shoot ratio, No of leaves and fresh weight, and nutritional metabolites (carbohydrates and proteins) were studied at intervals of two weeks from 4th week onwards using un inoculated tomato seedlings as control.

Figure 6: Greenhouse studies of tomato seedlings treated with AVSW 1  Click here to view Figure

In previous findings, endophytic bacterial strains, namely CT12, CT 13 and CT 16 of S. maltophilia isolated from fruit, stem and leaf of tomatoes, respectively, showed nearly 35.9 % enhancement of shoot height and 19.6-28.3% root length of tomato seedlings and also observed enhancement of fresh weight of shoot and root by 39.5 -57 % and 38.2 -58.8 % respectively under greenhouse conditions ⁴¹.

Similarly, Stenotrophomonas maltophilia BJ01 isolated from halo tolerant grass species showed a 17.68 % enhancement of shoot height  and 57.14 % enhancement of fresh weight in peanut plants compared to control under high salt stress conditions  (100 mM  NaCl )⁹

In previous findings, Stenotrophomonas maltophilia SBP-9 rhizospheric isolate of Sorghum bicolour showed significant enhancement of 20 % shoot height, 28.81% root length, 28 % fresh weight and 42 % dry weight of the Plant and 18.40 % shoot fresh weight. It also reported that chlorophyll content increased by 55% at 150 mM  NaCl, 39 % at 200 mM NaCl and 25 % at 200 mM NaCl when compared with 0 % NaCl in wheat plants ⁶.

S.maltophilia JVB5 isolated from sunflower root endosphere showed significant enhancement of root length ( 30.23 %), lateral roots (37.45 % ) and fresh weight (31.65%). IPR-Pv696 and 262XG2, two Bacillus toyonensis strains of root nodules of clover, showed a significant increase in fresh weight to (40.53 fed ^-1 and 42.68 tons fed ^-1), chlorophyll content by 4.51 % and carbohydrate content by 1.519 %, 20.18 % respectively ⁴ .

Similarly, in the present investigation, AVSW 1 treated tomato seedlings showed significant response in terms of the growth attributes and nutritive metabolites from the fourth week to the 8th (fig5.1 to 5.3). A significant increase of Shoot length (53.47 % ,41.85 % and 40.55 %) root length( 48.97 % , 48.76%  and 52.97 %), number of leaves (92.29 %, 69.07 %, 84.33 %), lateral roots (52.81 %,43.77 %, 37.08 % ), fresh weight of Plant (90.10 %, 99.60 %, 81.6 %) in 4^th, 6^th and 8^th weeks respectively in AVSW 1 treated plants compared to control (Fig 6 ). AVSW 1 treated plants also showed significant enhancement of protein and carbohydrate (66.66 %, 43.83 %, 45.75 % & 33.33 %, 33.96 %, 29.50 %) from 4^th week to 8^th week . Our results emphasized that the isolate AVSW 1 can promote plant growth and nutritional health and its antifungal activity against Fusarium oxysporum.

Based on the study, there is a significant increase in root and shoot length, number of leaves and lateral roots when compared withcontrol, protein and carbohydrate content and fresh weight is more in inoculated seeds and seeds treated with AVSW 1 inoculum showed better antifungal activity against  Fusarium  oxysporum .

Figure 7A: Root length and shoot height   on 4th ,6th and 8th week in treated and control plant Click here to view Figure

Figure 7B: Fresh weight and number of leaves on 4th, 6th and 8th week in  treated and control plants. Click here to view Figure

Figure 7C: Lateral roots, Protein and carbohydrate on 4th, 6th and 8th week in  treated and control Click here to view Figure

Figure 8: FTIR spectral analysis and interpretation of bioactive metabolites of Sphenotrophomonas maltophila  AVSW1. Click here to view Figure

Table 3: FTIR Spectral Data Analysis of Stenotrophomonas maltophila

Wavelength range	Functional groups and their bonds
3314.25	C-H stretch, Alkyne (strong, sharp)
3303.73	OH stretch normal polymeric
2944.98	C-H stretch, Alkane (medium)
2832.35	N-H stretch, Alcohol (weak, broad)
2042.92	N=C=N stretch, Isothiocyanate (strong)
1659.67	C=C stretch, Alkene (medium)
1449.21	C-H bend, Alkane (medium)
1414.57	S=O stretch, Sulfate (strong)
1114.16	C-O stretch, secondary alcohol (strong)
1019.84	C-F stretch, Fluoro compound (strong)

 In GC MS chromatogram major peak was observed at 6 min 8sec with the retention factor value of 43.00 with the molecular weight of 42.9582g/mol with the chemical formula C6H12O2.Compared to the NIST library, the primary compound was identified as 2-pentanone, 4-hydroxy, 4-methyl ester, which is confirmed by the FTIR results. Similarly, the compounds identified in the GC-MS analysis are confirmed to be the same as those in the FTIR analysis. As per the results of GC-MS, the profile of the AVSW 1 extract contains volatile organic compounds and secondary metabolites, which can efficiently control phytopathogens, increase plant growth, and induce systemic resistance^37-39.

Figure 9: GC-MS analysis  of metabolites of Stenotrophomonas  maltophila  AVSW1 at RT 6.30 GC-MS  Chromatogram of Stenotrophomonas  maltophila AVSW 1. Click here to view Figure

 Table 4: Metabolic Profile of  Bacterial extract of  AVSW 1  Stenotophomonas  maltophilia

S.No	RT	Height	Name of the compound	Molecular Formula	Mol wt	Area  % occupied	Norm %
1	6.830	30,461,028	2-Pentanone , 4-Hydroxy-4-Methyl-	C6H12O2	116	20.389	100
3	19.990	44,1313,68	Cyclopropane,1-(1-Methyethyl)-2-Nonyl-	C15H30	210	15.419	19.53
3	19.050	216,875,040	Glycine, N-Acetyl-N-(Trifluoro acetyl)-, Methyl Ester	C7H8F3NO4	227	13.917	18.88
4	17.559	106,691,120	2-Acetoxy  Iso butyryl Chloride	C6H9O3Cl	164	13.507	6.54
5	17.274	40,798,592	Propanoic Acid, 2-Oxo-, Methyl Ester	C4H6O3	102	12.275	60.20
6	18.500	52,147,852	Pentanoic Acid, 4-Oxo-	C5H8O3	116	7.883	66.24
7	7.395	20,769,012	2-Pentanone, 4-Hydroxy-4-Methyl-	C6H12O2	116	3.983	38.66
8	7.660	15,587,187	2-Hexanone, 6-Acetyloxy)	C8H14O3	158	3.849	16.79
9	18.640	35,413,672	5-Hydroxy pentane hydroxyl amine, N,O,OTriacetyl-	C11H19O5n	245	3.423	68.26
10	22.727	42,736,244	Ethanol, 2-(Octyloxy)-	C10H22O2	174	2.664	6.66
11	19.330	24,171,646	2-yclopenten-1-One, 2-Hydroxy -3,4-Dimethyl-	C7H10O2	126	1.357	75.62
12	16.194	13,718,134	2-Pentanone, 4-Hydroxy-4-Methyl-	C6H12O2	116	1.333	13.06

Figure 10: GC-MS spectrum of Active metabolites of Sphenotrophomonas matalopjila AVSW 1 at RT 6.830 Click here to view Figure

Metabolic Profile of Bacterial Extract of AVSW 1 Stenotrophomonas maltophilia  

As per the NIST Database, bioactive metabolites such as 2-Pentanone, 4-Hydroxy-4-methyl, Cyclopropane,1-(1-Methyl ethyl)-2-Nonyl-, Glycine, N-Acetyl-N-(Trifluoro acetyl)-, Methyl Ester, 2-Acetoxy Iso butyryl  Chloride, Propanoic Acid, 2-Oxo-, Methyl Ester, Pentanoic Acid, 4-Oxo-,5-Hydroxypentanehydroxylamine, Ethanol, 2-(Octyloxy)-, 2-Cyclopenten-1-One, 2-Hydroxy-3,4-Dimethyl- , 2,2- Di methyl tetra hydro pyran-4-ol showed bioactivities like root colonization, antifungal, high salinity stress tolerance, production of secondary metabolites and agrochemicals, signalling of molecules, enhanced microbial tolerance, anti-oxidation, cryoprotection, immune response, regulating  bacterial glycogen metabolism, gluconeogenesis, anti-inflammatory, plant defence mechanisms and promotes plant growth and metabolism

In earlier studies, Stenotrophomonas maltophilia UN1512 of strawberries reported the presence of Benzothiazole, 1,3,5,7-Cyclooctatetraene-1-carboxaldehyde, Carbonic Acid, octadecyl phenylester,  Benzaldehyde,2,5-bis[(trimethylsilyl)oxy]-, Estragole, and Benzaldehyd , and significantly inhibited the growth of fungal pathogens and promote growth of tomato plants in vitro ⁴³. 

Volatile compounds like haloalkanetrichloromethane, 2,4-dimethyl heptane and 4-methyl octane, furans and dimethyl sulphide, α- and β-pinene, camphene, and Δ-3-carene were reported in S.rhizophila EP2.2 which are potential antifungal metabolites against A.alternata and B.cinerea^{18 ,44-47,49,50}

Discussion

Currently, utilizing PGP bacteria in agriculture as Bio fertilizers and Bio inoculants is more winsome. Such bio inoculants protect plants from diseases and biotic and abiotic stress conditions by producing various regulatory chemicals, volatile organic compounds and secondary metabolites in the vicinity of the rhizosphere. Many Bacterial endophytes can colonize an ecological niche, making them potential biocontrol agents against diseases.

Phosphate solubilising endophytic bioinoculant is one of the alternative biotechnological solutions in sustainable agriculture to meet the phosphate demands of plants. IAA is one of the most crucial signal molecules in regulating plant growth. Salinity, pH and temperature are primordial abiotic factors which control plant growth, photosynthetic capacity, protein synthesis, energy, lipid metabolism, and total nitrogen content. PGP bacterial isolates which can have acclimatization to extreme high and low levels of salinity, pH and temperature will be a potential bioresource for sustainable crop productivity. In present research the isolate, Stenotrophomonas maltophilia  AVSW 1, showed multiple potential by having acclimatization to abiotic factors apart from direct (IAA, phosphate solubilisation, Ammonia) and indirect  (HCN, volatile compounds, siderophore s) mechanisms. Secondary metabolites such as 2- Pentanone, 4-Hydroxy-4-methyl, Cyclopropane,1-(1-Methyl ethyl)-2-Nonyl-, Glycine, N-Acetyl-N-(Trifluoro  acetyl)-, Methyl Ester, 2-Acetoxy Iso butyryl Chloride, Propanoic Acid, 2-Oxo-, Methyl Ester, Pentanoic Acid, 4-Oxo-,5-Hydroxypentanehydroxylamine, Ethanol, 2-(Octyloxy)-, 2-Cyclopenten-1-One, 2-Hydroxy-3,4-Dimethyl-, 2,2- Di methyl tetrahydro pyran-4-ol present in AVSW 1 are reported to be responsible for root colonization and suppression of soil borne fungal pathogens. Hence tomato plants inoculated with AVSW1 showed enhanced growth in greenhouse trials. Further advanced technological intervention can be useful to bring potential formulation with this isolate Stenotrophomonas maltophila AVSW1.

Conclusion

In conclusion, Tomato plants inoculated with AVSW 1, an endophytic plant growth promoting bacteria isolated from chill roots effectively suppressed fungal (Fusarium oxysporum) growth and exhibited significant growth-promoting traits and growth enhancement compared to untreated control. In this study, PGP isolate AVSW 1 showed maximum PGP traits in in-vitro conditions, perhaps can be used as a plant growth promoter in several vegetable or fruit crops. AVSW 1 improves plant growth directly through phosphate solubilization, production of secondary metabolites, volatile organic compounds, and growth hormones including nitrogen fixation. It reduces phytopathogens by sequestration of iron, secretion of siderophores, release of volatiles, etc. Further, the growth of plant pathogens may be directly inhibited by antibiosis. AVSW 1 may also induce systemic resistance in the host, where the plant defence mechanism gets activated against pathogen attack. The results indicate that Plant growth-promoting rhizobacteria AVSW 1 isolated from chilli roots can help the tomato plant withstand stress and support the Plant morphologically, physiologically, and biochemically. It can potentially promote tomato growth directly or indirectly, and further exploration of AVSW 1 as a bioinoculant can be tested at the field level to confirm its commercial significance. Genomic, proteomic and meta bolomics of holobiome (Plant and associated micro biome) and interdisciplinary research findings of AVSW 1 will be beneficial to understand its biocontrol potential, mode of action, regulatory mechanisms, and plant-microbe interaction in detail.

Acknowledgement

The authors are thankful to the Acharya Nagarjuna Univeristy, Guntur, A.P for providing the well-equipped Laboratories along with required infrastructure to complete the study.

Funding Sources

Authors are thankful to the DST-FIST Instrumentation centre(DST-Delhi SR/FST/LS-1/620/2015) of Dept of Botany and Microbiology of Acharya Nagarjuna University, Guntur, Andhra Pradesh, India and the corresponding author thankful to University Grants commission for providing financial assistance of UGC-MRP (F.No 40-132/201(SR)).

Conflict of Interest

The authors declare that there is no conflict of interest

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal, subjects, or any material that requires The ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Authors’ Contribution

Gadala Swapna carried out the laboratory work, conceptualization, wrote the manuscript text and methodology. Amrutha V. Audipudi was the supervisor of the research work

References

1. Alori E.T., and  Babalola O.O. Microbial Inoculants For  Improving Crop Quality and  Human Health in Africa. Frontiers In Microbiology, 2018 ; 9:1-11.Doi.Org/10.3389/Fmicb.2018.02213
2. Abdallah  R.A.B., Khiareddine  H.J., Nefzi  A., and  Remadi  M.D. Evaluation  of the Growth- Promoting Potential of  Endophytic Bacteria Recovered  from  Healthy Tomato Plants. Journal Of Horticulture , 2018; 5(2): 1-10. Doi: 10.4172/2376- 0354.1000234
3. Saikia B., Gogoi S., Ajit Kumar Savani A.K., and Bhattacharyya  A. Metabolites and Peptides of Endophytic Origin  in Plant Growth Promotion  and Defence  Reactions  in Solanaceous Crop Tomato .New And Future Developments In Microbial Biotechnology And Bioengineering, 2022; 89-110. Doi.Org/10.1016/B978-0-323-85579-2.00005-8
4. Adeleke  B.S., Ayangbenro A.S., and Babalola O.O. Effect Of Endophytic Bacterium, Stenotrophomonas maltophilia  JVB5 on  Sunflowers . Plant  Protectio Science ,2022; 58(3) :185-198. Doi: 10.17221/171/2021-PPS
   CrossRef
5. Alexander A., Singh V.K., Mishra A., and Jha B. Plant Growth Promoting Rhizo Bacterium Stenotrophomonas  maltophilia  BJ01 Augments Endurance Against N2 Starvation By Modulating Physiology and  Biochemical Activity of  Arachis  hypogea. 2019;14(9):1-20. Doi.Org/10.1371/Journal.Pone.0222405
6. Singh R.P.and Jha P.N.The PGPR  Stenotrophomonas maltophilia SBP-9 Augments Resistance Against Biotic and Abiotic Stress in Wheat Plants. Front Microbiol, 2017;8:1-15.Doi:10.3389/Fmicb.2017.01945
   CrossRef
7. Kumar A., Singh V.K., Tripathi V., Singh  P.S., and  Singh A.K. Plant Growth-Promoting rhizobacteria (PGPR): Perspective in Agriculture Under Biotic and Abiotic   Stress .Crop Improvement Through Microbial Biotechnology, 2018; 333-  342 Doi.Org/10.1016/B978-0-444-63987-5.00016-5
8. Singh V. K., Singh A.K., Singh P.P., and  Kumar A. Interaction  of Plant Growth Promoting Bacteria With Tomato Under Abiotic Stress: A  Review. Magriculture, Ecosystems & Environment, 2018;  267 : 129-140.Doi.Org/10.1016/J.Agee.2018.08.020
   CrossRef
9. Alexander A.,Singh V.K., and Mishra  A.Halotolerant PGPR  Stenotrophomonas maltophilia BJ01 induces Salt Tolerance by Modulating Physiology And Biochemical Activities of  Arachis hypogaea. Front microbiol , 2020;14 (9):1-20. Doi: 10.3389/Fmicb.2020.568289
   CrossRef
10. Ulrich,K., Kube M.,Becker R.,Schneck V.,UlrichA. Genomic Analysis of  the Endophytic Stenotrophomonas Strain 169 Reveals Features  Related To Plant- Growth Promotion  and  Stress Tolerance:Sec.Microbe and Virus Interactions With Plants . Front. Microbiol., 2021;12:1-14 .Doi.Org/10.3389/Fmicb.2021.687463
11. Osman  N. H.,  and Ali A. Isolation, Identification And Evaluation of The Plant Growth Promoting Activities of Endophytic Stenotrophomonas maltophilia  to Stimulate Growth of  Clover  Plants Under Salt Stress. Microbiology Research Journal International,2022 ; 32(8): 7-20.  Doi: 10.9734/Mrji/2022/V32i81336
    CrossRef
12. Aeron A., Dubey R.C., and  Maheshwari D.K.Characterization  of a Plant-Growth-Promoting Non-Nodulating endophytic Bacterium (Stenotrophomonas maltophilia) From the Root Nodules of  Mucuna utilisVar. Capitata L.(Safed kaunch) . Canadian Journal Of Microbiology , 2020;66(11):1-9. Doi.Org/10.1139/Cjm-2020-0196
13. Aktas C.,Ruzgar  D., Gurkok S., and Gormez A.Purification  and  Characterization of  Stenotrophomonas Maltophilia   Chitinase   With Antifungal  and Insecticidal Properties.  Preparative Biochemistry& Biotechnology,2022; 53(7): 797-806.  Doi.Org/10.1080/ 10826068.2022.2142942
    CrossRef
14. Quan C.,Wang X., and  Fan S. Antifungal Compounds of Plant Growth Promoting Rhizobacteria and its Action Mode . Plant Growth And Health Promoting  Bacteria, 2010;18:117-156. Doi:  10.1007/S11274-017-2364-9
    CrossRef
15. Deshmukh S. K., Gupta M.K., PrakashV., And Saxena S. Endophytic Fungi: A Source of Potential Antifungal Compounds. Journal Of Fungi , 2018;4(3):77. Doi.Org/10.3390/Jof4030077
    CrossRef
16. Ali S. Brilli ., Hameed S., Shahid M. Iqbal M.,Lazarovits G.,and Imran A. Functional Characterization of Potential PGPR Exhibiting Broad-Spectrum Antifungal Activity . Microbiol Res , 2020;232(126389) :1-1. Doi.Org/10.1016/J.Micres.2019.126389
    CrossRef
17. Raio A., Brilli F., Neri L., Baraldi R., Orlando F., Pugliesi, Chen  X., And  Baccelli  I. Stenotrophomonas  rhizophila Ep2. 2  Inhibits Growth of  Through the Emission of Volatile Organic Compounds, Botrytis Cinerea  Restricts Leaf Infection  And Primes Defence Genes. Frontiers In Plant Science, 2023;14:1-17. Doi:10.3389/Fpls.2023.1235669
    CrossRef
18. Solís D.R., Salmón E. Z., Pérez  M.C., Granados M.C.R., Rodríguez  LM., and Santoyo G. Pseudomonas  Stutzeri   E25  and Stenotrophomonas maltophilia CR71 Endophytes Produce Antifungal  Volatile   Organic  Compounds and Exhibit Additive Plant Growth-Promoting  Effects.  Biocatalysis And   Agricultural Biotechnology , 2018;13:46-52.  Doi.Org/10.1016/J.Bcab.2017.11.007.
19. Alijani  Z., Amini J., Ashengroph M., and Bahramnejad B.Volatile Compounds Mediated Effects of  Stenotrophomonas  maltophilia Strain UN1512  in Plant Growth Promotion  and its Potential For the Biocontrol of  Colletotrichum  nymphaea . Physiological  And Molecular Plant Pathology, 2020;112:1-13. Doi.Org/10.1016/J.Pmpp.2020.1015552020
20. Hisatomi​ A.,Shiwa​ Y.,Fujita​ N., Koshino​ H.,Tanaka​ N.Identification and Structural Characterization  of a  Catecholate-Type  Siderophore  Produced By  Stenotrophomonas maltophilia  K279a .Microbiology Society, 2021; 167(7). Doi.Org/10.1099/Mic.0.00107
21. Vacheron  J.;  Desbrosses G.; Bouffaud M.L.; Touraine B.; Moënne-Loccoz  Y.; Muller D.; Legendre  L.; Wisniewski-Dyé F.; Prigent-Combaret C. Plant Growth-Promoting  Rhizobacteria and Root System Functioning. Frontiers In Plant Science, 2013;4: (356);1-19. Doi.Org/10.3389/Fpls.2013.00356
22. Mahdi O., Eklund B., And  Fisher N.Stenotrophomonas maltophilia:  Laboratory Culture  and Maintenance. Current Protocols In Microbiology , 2014 ;32:6F.1.1- 6F.1.6. Doi.Org/10.1002/9780471729259.Mc06f01s32
23. Cochard  B., Giroud B., Crovadore  j.,Chablais  R.,Arminjon  L., and Lefort F., Endophytic PGPR From Tomato Roots: Isolation, In Vitro  Characterization and InVivo Evaluation of Treated Tomatoes (Solanum  lycopersicum  L.) . Microorganisms, 2022;10(4),765.  Doi.Org/10.3390/ Microorganisms10040765
24. Kumar N.R.,V. Thirumalaiarasu., And  P. Gunasekaran. Genotyping of  Antifungal Compounds Producing Plant Growth-Promoting Rhizobacteria  Pseudomonas Fluorescens. Current Science , 2002;82(12):1463-1466
    CrossRef
25. Allu S., N. Pradeep Kumar, And  Audipudi  A.V. Isolation, Biochemical and   PGP Characterization of Endophytic Pseudomonas aeruginosa Isolated From Chilli Red  Fruit  Antagonistic Against Chilli Anthracnose Disease.  International Journal Of Current  Microbiology And Applied Sciences, 2014; 3(2) : 318-329.
26. Sarbadhikary S., And Mandal  N.C.  Field Application of Two  Plant Growth Promoting  Rhizobacteria With Potent Antifungal Properties.  Rhizosphere , 2017;3(1). : 170-175. Doi.Org/10.1016/J.Rhisph. 2017.04.014.
    CrossRef
27. Patten C.L., Glick B.R. Role of Pseudomonas  putida  indole-Acetic Acid  in Developing the Host Plant Root System.  Appl. Environ. Microbiol, 2002;68(8):3795-DOI: https://doi.org/10.1128/AEM.68.8.3795-3801.2002
    CrossRef
28. Husen  E. Screening of Soil Bacteria For Plant Growth-Promoting Activities  in Vitro. Indones. J. Agric. Sci, 2003;4(1):27- 31. DOI:10.21082/Ijas.V4n1.2003.27-31
    CrossRef
29. Aneja K. R. Experiments In Microbiology, Plant Pathology And Biotechnology. New Age International Publishers, India , 2007.
30. Desire H.M., Bernard F., Forsah  M.R., Assang C.T., And Denis O.N. Enzymes And Qualitative Phytochemical  Screening of Endophytic Fungi Isolated From Lantana Camara Linn. Leaves. Journal of  Applied Biology  and Biotechnology , 2014; 2(6): 001-006. DOI: 10.7324/JABB.2014.2601
    CrossRef
31. Maki L.M., Broere M., Leung, K.T., and Qin W. Characterization of Some Efficient Cellulase- Producing Bacteria Isolated From Paper Mill  Sludges and  Organic Fertilizers.  Int J Biochem  Mol Biol,  2011;2(2):146-154.
32. Cappuccino J. G., And N. Sherman..Biochemical Activities of Microorganisms. Microbiology, A Laboratory Manual. The Benjamin/Cummings Publishing Co. California,  USA, 1992: 188-247.
33. Holt J.G., Krieg N.R., Sneath P.H.A., Staley J.T., And  Williams S.T. Bergey’s Manual of  Determinative Bacteriology, 9th Ed.Williams And Wilkins, Baltimore, USA.
34. Saitou N., and Nei  M.The Neighbour-Joining Method: A New Method For Reconstructing Phylogenetic  Trees . Molecular Biology And Evolution,  1987;4(4): 406– 425. Doi.Org/10.1093/Oxfordjournals.Molbev.A040454
35. Bhutto M.A., Dahot and M. Umar. Effect of Alternative Carbon and  Nitrogen Sources on Production of Alpha-Amylase  by Bacillus Megaterium. World   Applied Sciences  Journal (Special Issue Of Biotechnology & Genetic Engineering),  2010; 8(2): 85- 90. Doi:10.1016/J.Jbiotec.2008.07.1864
    CrossRef
36. Maity J.P.,Kar S.,Lin C.L.,Chen Y.C.,Chang Y.C.,Jean S.J., and KulpT.K. Identification and Discrimination of Bacteria Using  Fourier Transform Infrared Spectroscopy. Spectrochimica Acta  Part A: Molecular And Biomolecular Spectroscopy, 2013;116:478-484.Doi.Org/10.1016/J.Saa.2013.07.062
    CrossRef
37. Ramzan M., Raza  A., Musharaff S.G.,UnNisa Z.Recent Studies On  Advance Spectroscopic Techniques For  Identifying   Microorganisms: A Review. Arabian Journal  Of Chemistry,  2023; 16 (3):104521.Doi.Org/10.1016/J.Arabjc.2022.104521.
    CrossRef
38. Haron F.K., Shah M.D.,Yong Y.S., Tan J.K., Lal M.T.M., And  Maran  B.A.V. Antiparasitic  Potential of Methanol Extract of   Brown Alga  Sargassum polycystum (Phaeophyceae) and  its  LC-MS/MS Metabolite Profiling. Diversity 2022; 14(10) 796; 1-12. Doi.Org/10.3390/D14100796
39. Moldoveanu , S.C., and  David  V. Derivatization  Methods  in GC And GC/MS. Intechopen, 2018; DOI:10.5772/Intechopen.81954
    CrossRef
40. Kumar N.P., and Audipudi, A.V. Exploration  of a Novel Plant Growth Promoting Bacteria  Stenotrophomonas  maltophilia AVP 27 Isolated  From the Chilli  Rhizosphere Soil.  International Journal Of Engineering Research And General Science ,2015;3(1): 265- 276; ISSN 2091-2730.        http://Www.Pnrsolution.Org/Datacenter/Vol3/Issue1/34.Pdf
41. Abdallah R.A.B.,Tlili S.M., Nfzi A.,Khiareddine H.J., and Remadi M,D. Biocontrol of Fusarium Wilt and Growth Promotion of Tomato Plants Using  Endophytic Bacteria Isolated From Nicotiana  glauca  Organs. Biological Control, 2016;97 : 80-88. https://Doi.Org/10.1016  /J.Biocontrol.2016.03.005
    CrossRef
42. Barthlomew  D.C.,Banin L.F.,Bittencourt P.R.L.,Suis M.A.F.,Mercado  L.M.,Nilus  R. Burslem D.F.R.P., and Rowland L. Differential Nutrient Limitation and Tree Height Control Leaf Physiology, Supporting Niche Partitioning in Tropical Dipterocarp Forests Functional Ecology , 2022; 36:2084-2103. DOI:10.1111/1365-2435.14094
    CrossRef
43. Alijani  Z., Amini J., Ashengroph M., and Bahramnejad B. Volatile Compounds Mediated Effects of Stenotrophomonas maltophilia Strain UN1512  in  Plant Growth Promotion and its Potential For The Biocontrol Of  Colletotrichum  Nymphaea. Physiological And Molecular Plant Pathology, 2020;112:101555 . Doi.Org/10.1016/J.Pmpp.2020.101555
    CrossRef
44. Kai M., Effmert U., Berg G., Piechulla B. Volatiles of  Bacterial Antagonists Inhibit  Mycelial Growth of the Plant Pathogen  Rhizoctonia solani. Arch. Microbiol, 2007;187(5) :351–360. Doi: 10.1007/S00203-006-0199-0 2007
    CrossRef
45. Cernava T., Aschenbrenner  I. A., Grube  M., Liebminger S., Berg G. A Novel Assay For the Detection of Bioactive Volatiles Evaluated  By Screening of Lichen Associated  Bacteria.  Front. Microbiol, 2015;6:398;1-9. Doi: 10.3389/Fmicb.2015.00398
    CrossRef
46. Reyes-Perez  J. J., Hernandez-Montiel  L. G.,Vero  S., Noa-Carrazana  J. C., Quiñones- Aguilar E. E., Rincón- Enríquez G. Postharvest  Biocontrol  of Colletotrichum gloeosporioides  on Mango Using the Marine Bacterium  Stenotrophomonas rhizophila  and  its Possible Mechanisms of Action .J. Food Sci. Technol, 2019;56, 4992– 4999. Doi: 10.1007/S13197-019-03971-8
    CrossRef
47. Rivas-Garcia T.,Murillo-Amador B., Nieto-Garibay A., Rincon-Enriquez G., Chiquito-Contreras R. G., Hernandez-Montiel L.G. Enhanced Biocontrol  of  Fruit Rot on Muskmelon  By Combination Treatment With Marine Debaryomyces hansenii  and Stenotrophomonas  rhizophila and their Potential Modes of Action . Postharvest Biol. Technol, 2019: 151;61–67.  Doi: 10.1016/J.Postharvbio.2019.01.013
    CrossRef
48. Lin Y.T., Lee  C.C.,Leu W.M.,Wu  J.J., Huang Y.C., Meng M. Fungicidal  Activity of Volatile  Organic Compounds Emitted by Burkholderia  gladioli Strain BBB-01. Molecules,2021 ;26(3):745: 1-15; Doi: 10.3390/molecules26030745
    CrossRef
49. Baerlocher  F.J., Langler  R.F., Frederiksen M. U., Georges N. M.,Witherell  R. D. Structure–Activity Relationships For Selected Sulfur-Rich Antifungal Compounds . Aust. J. Chem, 1999;52:167–172. Doi: 10.1071/C98141
    CrossRef
50. Groenhagen U., Baumgartner R., Bailly A., Gardiner A., Eberl  L., Schulz  S. Production of Bioactive Volatiles By Different Burkholderia  ambifaria  Strains. J. Ecol, 2013; 39: 892–906. Doi: 10.1007/S10886-013-0315-Y
    CrossRef
51. Wang  C., Wang Z., Qiao X., Li Z., Li F., Chen M. Antifungal Activity of  Volatile Organic Compounds From Streptomyces  alboflavus TD-1. FEMS .Microbiol. Lett., 2013; 34(1): 45–51.Doi: 10.1111/1574-6968.12088
    CrossRef
52. Li Q. L.,Ning  P., Zheng L., Huang  J. B., Li G. Q., Hsiang T. Fumigant Activity of  Volatiles of  Streptomyces  globisporus  JK-1 against  Penicillium  italicum on  Citrus microcarpa .  Postharvest Biol.Technol,  2010;58: 157–165. Doi:10.1016/J.Postharvbio.2010.06.003
    CrossRef
53. Avalos M., Garbeva P., Vader L., Van Wezel G. P., Dickschat J. S., Ulanova  D. Biosynthesis, Evolution And Ecology Of Microbial Terpenoids. Nat. Prod. Rep, 2022;39: 249–272. Doi: 10.1039/D1np00047k
    CrossRef
54. Song  C., Schmidt  R., De Jager V., Krzyzanowska D., Jongedijk E., Cankar  K. Exploring the Genomic Traits Of Fungus-Feeding Bacterial Genus Collimonas . BMC Genomics 2015:16:1103. Doi: 10.1186/S12864-015-2289-3
    CrossRef