Physiological and Biochemical Attributes of an Endophyte Stenotrophom*onas maltophila, AVSW 1 Isolated from Chilli on   PGP of Tomato (2024)

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 Phosphates , 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, Stenotrophom*onas maltophilia isolate, extracted from green fruits of chilli and its physiological attributes in the growth promotion of tomato.

Isolation ofStenotrophom*onas 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 Methodology

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°Cfor 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 ofbacterial 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 againstFusarium 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 ofS. maltophiliaAVSW 1 showed 4-8 h of Lag phase,12-44 h of Exponential phase, and 44-68 h ofstationary phase. After 68 h, the decline phase was observed (Fig2).

Figure 1: Optimization of Physical and Chemical parameters ongrowth 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. maltophiliaAVSW 1,IAA production increased by 55.66 %,Ammonia production increased by74.85 % ,Phosphate (po4^– ) solubilization increased by 69.42% and Siderophore production increased by 49.62 % after Optimization.Whereas in earlier findings,S. maltophiliaJVB5, root endophyte of sunflower showed optimization were IAA(30.0 μg/ml) ,Phosphate solubilisation (32.23 ppm) and Siderophore (79.90psu ) after optimization ⁴.Previous findings reported that two S.maltophila IPR-Pv696 and 262XG2 root nodular endophyte strains of clover reported IAA (30.26μg /ml and 31.15 μg/ml), Phosphate solubilization (100 and 75%) and phosphate liberated (278 mg/land 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 inS. maltophilia ,SBP-9 rhizobacteria ofSorghum bicolour at pH 8, temperature 30 ⁰ C,Nacl Concentration 6 % and broth incubated for 48 h ⁶.818 ppm Phosphatesolubilisation, 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 Stenotrophom*onasmaltophiliaAVP 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.

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 asStenotrophom*onasmaltophiliaand deposited in NCBI GENBANK with the nameStenotrophom*onasmaltophiliaAVSW 1with the accession noOQ293900

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

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

Greenhouse studies

The plant growth promotion potential of Stenotrophom*onas maltophiliaAVSW 1was 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 onwardsusing 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. maltophiliaisolated 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 weightof shoot and root by 39.5 -57 % and 38.2 -58.8% respectively under greenhouse conditions ⁴¹.

Similarly,Stenotrophom*onas maltophilia BJ01isolated 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,Stenotrophom*onas maltophilia SBP-9 rhizospheric isolate ofSorghum 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 % NaClin wheat plants ⁶.

S.maltophilia JVB5 isolated from sunflower root endosphereshowed significant enhancement of root length ( 30.23 %), lateral roots (37.45 % ) and fresh weight (31.65%). IPR-Pv696 and 262XG2, twoBacillus toyonensisstrains 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). Asignificant 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 againstFusarium oxysporum.

Based on the study, there is a significant increase in root and shoot length, number of leaves and lateral roots when compared with control , protein and carbohydrate content and fresh weight is more in inoculated seeds and seeds treated with AVSW 1 inoculum showed better antifungal activity againstFusarium 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 Sphenotrophom*onas maltophila AVSW1. Click here to view Figure

Table 3: FTIR Spectral Data Analysis of Stenotrophom*onas maltophila

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 Stenotrophom*onas maltophila AVSW1 at RT 6.30 GC-MS Chromatogram of Stenotrophom*onas maltophila AVSW 1. Click here to view Figure

Table 4: Metabolic Profile of Bacterial extract of AVSW 1 Stenotophom*onas maltophilia

Figure 10: GC-MS spectrum of Active metabolites of Sphenotrophom*onas maltophilaAVSW 1 at RT 6.830 Click here to view Figure

Metabolic Profile of Bacterial Extract of AVSW 1 Stenotrophom*onas 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 showedbioactivities 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 , Stenotrophom*onas 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 Benzaldehyde , and significantly inhibited the growth of fungal pathogens and promote growth of tomato plants in vitro⁴³.

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, utilizingPGP 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 compoundsand 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 , Stenotrophom*onas 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 Stenotrophom*onas maltophila AVSW1.

Conclusion

In conclusion, Tomato plants inoculated with AVSW 1,an endophyticplant 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 hormonesincluding 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

The manuscript incorporates all datasets produced throughout the research study

Ethics Approval Statement

Not applicable

Author’s 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

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