Physiological and Molecular Plant Pathology 119 (2022) 101836

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Physiological and Molecular Plant Pathology

journal homepage: www.elsevier.com/locate/pmpp

Pseudomonas fluorescens VSMKU3054 mediated induced systemic resistance

in tomato against Ralstonia solanacearum
P. Suresh a, V. Shanmugaiah a, *, Rajakrishnan Rajagopal b, K. Muthusamy c, V. Ramamoorthy d
a
Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, Tamil Nadu, India
b
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia
c
Grassland and Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan, 31000, South Korea
d
Department of Plant Pathology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai, 625 104, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Keywords: Plant growth-promoting rhizobacteria (PGPR) are potential bio agents for the suppression of various soil-borne
Induced resistance diseases in crop plants and considered as viable alternative to chemical pesticides. The selected Pseudomonas
Ralstonia solanacearum fluorescens isolate VSMKU3054 is an effective biological control inoculum for the suppression of wilt disease of
Biocontrol
tomato incited by Ralstonia solanacearum by inducing plant defense enzymes viz., peroxidase (POX), polyphenol
Plant defense enzymes
oxidase (PPO), lipoxygenase (LOX), phenylalanine ammonia-lyase (PAL) and total phenol content. The highest
level of defense enzymes viz., POX, PAL, PPO, LOX and total phenol contents were observed in tomato seedlings
treated with P. fluorescens at 24h, 36h, 36h, 48h and 24h after challenge inoculation of R. solanacearum,
respectively. Whereas application of P. fluorescens alone in tomato plants also brought about induction of higher
levels of POX, PAL, LOX and total phenol contents, and a moderate level of PPO activity compared to pathogen
inoculated plants and control plants. The maximum induction of four prominent PPO isoforms was observed in
tomato plants upon the treatment of P. fluorescens VSMKU3054 and challenged with R. solanacearum. Thus, this
study showed that induction of defense-related genes by P. fluorescens plays an important role against
R. solanacearum.

1. Introduction importance and considered as a hopeful method to make permanent

solutions for disease management and enabling sustainable agriculture
Tomato wilt disease incited by Ralstonia solanacearum is one of the [8–11]. Numerous rhizosphere microbes such as Pseudomonas sp., Ba­
most destructive and economically significant disease [1,2]. It brought cillus sp., Streptomyces sp., Burkholderia sp. and Trichoderma sp. were
about 26% yield loss of fresh fruit production and yield loss may attain involved in Induced systemic resistance (ISR), biocontrol activity and
up to 91% under severe disease incidence [3]. In India, it is a major plant growth promotions [7,12]. Pseudomonas sp. is a well-known po­
constraint for tomato production. Pesticides such as copper-based fun­ tential biological control agent for the suppression of various phyto­
gicides, streptomycin sulphate, and bleaching powders were previously pathogens, and it also demonstrated significant plant growth-promoting
used to control tomato soil wilt disease [4,5]. But the chemical pesticide activity through the production of antimicrobial compounds such as 2,
causes unadorned hazards to the environment and public health [6,7]. 4-diacetylphloroglucinol, phenazine-1-carboxamide, hydrogen cya­
Further, the use of chemical fungicides in agriculture system for the nide, siderophore, lytic enzymes, ISR mediated enzymes and plant
management of plant diseases increase the cost of cultivation and also growth hormones like IAA [2,8,13,14].
create pesticide residual problem in our environment. Induced Systemic Resistance (ISR) refers to the development of self-
As a result, there is an urgent need to develop alternative approaches protective capacity in crop plants against various pathogens after
for controlling tomato wilt disease without causing hazards to envi­ appropriate priming with non-pathogenic organisms, particularly
ronment. In recent days, the biological control method is gaining saprophytic rhizosphere bacteria and fungi. Beneficial microbes

Abbreviations: PGPR, Plant growth-promoting rhizobacteria; ISR, Induced Systemic Resistance; PBS, Phosphate buffer saline; OD, Optical Density; CFU, Colony
forming unit; mM, milli molar; μl, Microliter.
* Corresponding author.
E-mail addresses: [email protected], [email protected] (V. Shanmugaiah).

https://doi.org/10.1016/j.pmpp.2022.101836
Received 5 December 2021; Received in revised form 27 March 2022; Accepted 4 April 2022
Available online 10 April 2022
0885-5765/© 2022 Elsevier Ltd. All rights reserved.
P. Suresh et al. Physiological and Molecular Plant Pathology 119 (2022) 101836

including various PGPR trigger systemic resistance against various dis­ followed by three washes with sterile distilled water for 3 min and blot
eases and insect pests. ISR by different PGPR is one of the significant dried. Further, they were soaked in 20 ml suspension of P. fluorescens (1
mechanisms in plant protection systems against various fungal, bacterial × 108 CFU ml− 1) culture amended with 0.2% carboxymethyl cellulose
and viral pathogens by enhancing plant immunity [15,16]. A recent (CMC) to facilitate adherence of bacterial cells on the tomato seeds and
report confirmed that ISR seems to be the outcome of numerous mech­ then incubated for 12h at 24 ± 2 ◦ C in a rotary shaker at 120 rpm. After
anisms and they are highly effective against various pathogens. Both incubation, the seeds were air dried aseptically [33]. Tomato seeds
induced resistance and responses were well-described in a wide range of soaked in sterile water containing 0.2% CMC served as control.
flowering plants [14,17]. ISR has been reported in a variety of plants In the second treatment, tomato seeds were pre-treated with
including tobacco, tomato, rice, sunflower, bean, carnations, cucumber P. fluorescens suspension (1 × 108 CFU) for 12h. They were germinated
and also the model plant Arabidopsis thaliana [4,7,16,18–20]. on moist blotter paper in petri dishes according to the International Seed
Rhizobacteria associated with host plants have also been found to Testing Association’s standard procedure (ISTA) (2005). The seeds were
increase the biosynthesis of defense-related enzymes within the plant incubated at 28 ± 2 ◦ C for 12 days until the seed leaf completely
system. Plant cells have advanced antioxidative pathways that involve emerged. The 12- day-old tomato seedlings were challenge-inoculated
the accumulation and activation of ISR related enzymes including LOX, with 20 ml of R. solanacearum suspension (1 × 108 CFU) [22] by root
PPO, POX, PAL, chitinase and β-1,3-glucanase. The production of anti­ dipping for 20 min and they were placed in moist filter paper in Petri
oxidant enzyme leads to cell wall thickening and structural deformation, dish (Fig. 1).
resulting in callose deposition and phenolic compound accumulation Tomato seedlings were inoculated with R. solanacearum alone in the
[19,21]. third treatment. Seedlings treated with PBS alone served as control.
Most of the horticulturally important crops especially solanaceous Tomato seedlings were harvested at 0, 12, 24, 36, 48, 60, and 72h after
crops viz., potato, tomato, chili and eggplants are severely affected by challenge-inoculation for analysis of defense-related proteins and
R. solanacearum. PGPR and Trichoderma spp. are well known biocontrol chemicals and the samples were stored at − 80 ◦ C [26]. The total protein
agents and implicated for the induction of various defense related en­ content was determined from the samples according to the Bradford
zymes and antimicrobial agents [19,22,23]. Pseudomonas sp.
VSMKU4050 and P. fluorescens DABBV4 as seed bacterization and soil
application improved seed germination and vigour index [24,25]. Pre­
vious research has revealed that P. fluorescens induced defense enzyme
activity significantly protected tomato plants from R. solanacearum [4,
13,22,26]. Pseudomonas sp. induced defense enzyme activity in different
plants such as mulberry [27], cucumber [28], banana [29,30] and rice
[7,20,31]. The objective of this study is to conduct a quantitative
analysis of defense-related proteins and to detect induced peroxidase
and polyphenol oxidase isoform patterns in P. fluorescens VSMKU3054
treated tomato plants challenge inoculated with R. solanacearum.

2. Materials and methods

2.1. Culture conditions

P. fluorescens VSMKU3054, a potential antagonistic isolate, was used

throughout the experiment. For mass multiplication, it was cultured on
King’s B agar medium at room temperature. Bacterial wilt pathogen
Ralstonia solanacearum was collected from the Indian Type Culture
Collection (ITCC), Indian Council for Agricultural Research (IARI), New
Delhi. It was grown on 2,3,5-Triphenyl Tetrazolium Chloride (TZC) agar
medium and further, it was sub-cultured in tryptic soya broth (TSB)
liquid culture medium. Both cultures were sub-cultured and maintained
in 30% glycerol at − 80 ◦ C for entire studies.

2.2. Preparation of bacterial inoculum

P. fluorescens was cultured in King’s B broth for 24 h at 37 ◦ C. After

incubation, cells were pelleted from the growth medium by centrifuging
the King’s B broth at 12,000 rpm for 10 min at 4 ◦ C. The cells were
cleaned with sterile 1X phosphate buffer saline (PBS) and they were
resuspended in PBS and adjusted to 1 × 108 CFU ml− 1 spectrophoto­
metrically at 600 nm. Similarly, R. solanacearum was grown overnight in
TSB medium. Bacterial cells were pelleted by centrifugation at 12,000
rpm for 10 min and further the pellets were dissolved in PBS. The cells
were adjusted to 1 × 108 CFU/ml− 1 spectrophotometrically (as one OD
approximately equals to 1 × 108 CFU/ml− 1) [22].

2.3. Tomato seed bacterization

Fig. 1. Effect of seed bacterization with P. fluorescens VSMKU3054 and chal­
The tomato seeds (variety PKM1) susceptible to bacterial wilt [32] lenge inoculated with R. solanacearum on tomato seedlings (PKM1). A – Control
were obtained from Horticultural College & Research Institute, Tamil (Untreated), B − P. fluorescens VSMKU3054, C − P. fluorescens VSMKU3054+
Nadu, India. They were surface sterilized with 1% sodium hypochlorite R. solanacearum, D – R. solanacearum.

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P. Suresh et al. Physiological and Molecular Plant Pathology 119 (2022) 101836

method. min. After incubation, the reaction mixture developed blue colour and
the absorbance was measured at 725 nm [35]. The activity was
2.4. Biochemical assessment of defense enzymes and chemicals in tomato measured as changes in absorbance and expressed as μg catechol− 1mg− 1
seedlings protein. For each treatment, three replicates were employed. The ex­
periments were carried out two times.
2.4.1. Estimation of POX
One gram of tomato seedlings was homogenized in 1 ml of 10 mM 2.4.6. Native PAGE analysis of peroxidase and polyphenol oxidase
phosphate buffer of pH 6.0. The homogenate was centrifuged at 12,000 Isoforms of POX and PPO were estimated by discontinuous native
rpm for 20 min at 4 ◦ C. The supernatant was used as an enzyme source polyacrylamide gel electrophoresis (Native-PAGE) according to
for the POX analysis. The POX assay was carried out with minor modi­ Laemmli (1970) [40] with minor modifications. The total proteins from
fications, as described by Hammerschmidt et al. [34]. The reaction tomato seedlings were extracted, subjected to non-denaturing condi­
mixture consisted of 0.25% (v/v) guaiacol in 10 mM potassium phos­ tions. 50 μl of crude extracts were mixed with loading dye bromophenol
phate buffer (pH 6.0) containing 10 mM hydrogen peroxide. To the re­ blue. Both treated and untreated samples were collected at 24 & 36h for
action mixture, 100 μl of crude enzyme extract was added into the same POX and PPO analysis respectively. Electrophoretic separation was
vial and incubated at room temperature. Changes in absorbance were carried out by following the standard procedure.
measured at 420 nm every 30 s for 3 min. Changes in absorbance min-1
mg-1 of protein were used to calculate enzyme activity. The experiments 2.4.7. POX and PPO staining method after electrophoresis
were carried out two times with three replications for each treatment The peroxidase isoform was detected by soaking the gels in a staining
[35]. solution prepared by dissolving 100 mg benzidine in 1 ml alcohol and
finally the volume was made up to 40 ml distilled water. 500 μl of glacial
2.4.2. Estimation of PAL acetic acid and 250 μl of H2O2 were added to the filtered solution, and
The modified Lisker et al. [36] procedure was used to assess PAL the gels were incubated in the solution until the blue band was visible
activity. One gram of tomato seedlings was macerated and homogenized [41]. PPO isoforms were determined by rotary shaking gels in 50 mM
with 1 ml of 25 mM Tris-HCL buffer (pH 8.8). The homogenate was Tris buffer (pH6.8) with 500 mg catechol and 300 mg L-3,4-dihydrox­
centrifuged at 10,000 rpm for 30 min at 4 ◦ C and the supernatant was yphenylalanine (L-DOPA) for 20 min until black bands appeared [37].
used for enzyme assay. The reaction mixture consisted of 1 ml enzyme
extract, 0.5 ml of 50 mM L-phenylalanine (substrate) and 0.4 ml of 25 2.4.8. Statistical analysis
mM Tris-HCL buffer (pH 8.8). The reaction was halted by adding 0.06 ml The ISR enzymes data were mean values of three replication and
of 5 N HCl to the reaction mixture after it had been incubated for 2h at analysed using the analysis of variance (ANOVA) and Duncan’s Multiple
40 ◦ C. The absorbance was measured at 290 nm against a blank reaction Range Test (DMRT). The data are expressed as mean values ± standard
mixture without L-phenylalanine. The rate of conversion of trans-­ deviation. Mean values with different letters differ significantly at p =
cinnamic acid (CA) from L-phenylalanine was used to determine enzyme 0.05 according to DMRT.
activity. For each treatment, three replicates were employed. The ex­
periments were carried out two times. The enzyme activity was 3. Results
expressed as mol of trans-cinnamic acid min− 1mg− 1protein [37].
3.1. Induction of resistance by P. fluorescens VSMKU3054 in tomato
2.4.3. Estimation of PPO against R. solanacearum
PPO activity was measured by the method of Mayer et al. [38]. 1 g of
tomato seedlings was homogenized in 10 mM phosphate buffer (pH 6.0). P. fluorescens VSMKU3054 remarkably induced and enhanced de­
The homogenate mixture was centrifuged at 12,000 rpm for 20 min at 4 fense capacity in terms of defense-related genes for the control of
◦
C and the supernatant was directly used as an enzyme source. 1.5 mL R. solanacearum because P. fluorescens VSMKU3054 has the potential to
0.1 M sodium phosphate buffer (pH 6.5) and 200 μl enzyme extract were generate substantial plant defense responses. Activating defense genes
used in the process. The reaction was started by adding 200 μl of 10 mM by PGPR is a unique technique in crop plant against biotic stress in
catechol to the mixture. For 1 min, the rate of catechol oxidation was addition to promoting plant growth and antibacterial activity. In this
measured at 420 nm. Changes in absorbance min− 1 mg− 1 protein were study, it is proposed that P. fluorescens VSMKU3054 colonizes on the
used to measure the activity. For each treatment, three replicates were root surface. The high level of defense elicitor molecules present on the
employed. The experiments were carried out two times [37]. surface of the antagonist could induce plant-associated defense enzymes
and pathogenesis-related proteins viz., PPO, PO, LOX, PAL and total
2.4.4. Estimation of lipoxygenase (LOX) phenol contents that are involved in protection against wilt disease of
The lipoxygenase activity was determined according to Borthakur tomato. Hence, we illustrate the process of ISR mediated by P. fluorescens
et al. [39]. One gram of tomato seedlings was homogenized with 0.2 M in tomato plants (Fig. 9).
sodium phosphate buffer (pH 6.5). The reaction mixture consisting 0.05
ml of the enzyme extract, 2.7 ml of 0.2 M sodium phosphate buffer (pH 3.2. Estimation of POX
6.5) and 0.3 ml of 10 mM linoleic acid in Tween 20. LOX activity was
measured by spectrophotometer and observed the appearance of con­ Following microbial actions, POX activity reached to the maximum
jugated diene hydroperoxide at 234 nm. Changes in absorbance min− 1 levels at 24h. POX activity was higher in tomato seedlings challenged
mg− 1 protein were used to measure activity [37]. For each treatment, with R. solanacearum than that were treated with P. fluorescens
three replicates were employed. The experiments were carried out two VSMKU3054 alone. However, POX activity in the tomato seedlings
times. treated with P. fluorescens VSMKU3054 alone remained at higher levels
(14.8/Δ 470 nm/min/mg protein) compared to untreated control.
2.4.5. Estimation of total phenol Whereas, the highest POX activity (23.1/Δ 470 nm/min/mg protein) was
One gram of tomato seedlings was homogenized with 80% methanol recorded in P. fluorescens VSMKU3054 treated tomato seedlings fol­
for 15 min. The homogenized tomato seedlings were centrifuged at lowed by challenged-inoculated with R. solanacearum (Fig. 2).
12,000 rpm for 20 min at 4 ◦ C. The reaction mixture consisted of 1 ml of
methanolic extract, 5 ml of sterile distilled water and 250 μl of Folin
Ciocalteau reagent (1 N). This solution was incubated at 25 ◦ C for 30

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P. Suresh et al. Physiological and Molecular Plant Pathology 119 (2022) 101836

3.5. Estimation of LOX

The maximum level of LOX activity was observed at higher levels in

P. fluorescens treated tomato seedlings challenge-inoculated with the
pathogen at 48h (49 Δ A234 nm/min/mg protein) after pathogen inoc­
ulation. Whereas moderate LOX activity (42.6 Δ A234 nm/min/mg
protein) was observed in tomato seedlings treated with P. fluorescens
alone. However, the low level of LOX activity (32.86 Δ A234 nm/min/mg
protein) was obtained in R. solanacearum alone treated tomato seedlings
(Fig. 5).

3.6. Total phenol content

Fig. 2. Peroxidase (POX) activity in tomato seedlings with different treatments. The maximum level of phenolic content (84.5 μg of catechol/min/
Control (Tomato seedlings treated with sterile distilled water); RS - (Tomato mg protein) was observed at 24h in tomato seedlings induced with
seedlings inoculated with R. solanacearum); P. fluorescens (Tomato seedlings P. fluorescens followed by challenge-inoculation with R. solanacearum.
inoculated with P. fluorescens VSMKU3054); P. fluorescens + RS (Tomato Tomato seedlings inoculated with the P. fluorescens VSMKU3054 alone
seedlings treated with P. fluorescens VSMKU3054 and challenge-inoculated with also recorded the higher level (69.16 μg of catechol/min/mg protein)
R. solanacearum. The letters above the bar indicate significantly different values
accumulation of phenolic content compared to R. solanacearum alone
(P = 0.05, DMRT analysis).
inoculated tomato seedlings and control seedlings (Fig. 6).

3.3. Estimation of PAL 4. Analysis of POX and PPO isozyme by native PAGE

P. fluorescens VSMKU3054 treated tomato seedlings challenge- The expression of POX was examined in protein samples of tomato
inoculated with R. solanacearum showed enhanced PAL activity. The seedling. POX1, POX2, POX3, and POX4 are four POX isoforms identi­
maximum PAL activity (41.07 μ.mol trans-cinnamic acid/min/mg pro­ fied by native PAGE analysis. The expression of isoforms POX3 and
tein) was observed at 36h after inoculation in P. fluorescens VSMKU3054 POX4 was higher in tomato seedlings inoculated with P. fluorescens and
pre-treated seedlings followed by challenge-inoculated with R. solanacearum. The intensity of the bands detected in tomato seedlings
R. solanacearum. Whereas, seedlings inoculated with R. solanacearum treated with P. fluorescens alone or that treated with R. solanacearum
alone showed lower PAL activity (29.73 μ.mol trans-cinnamic acid/min/ alone remained at lower levels. Protein extracts from the control plants
mg protein) and seedlings treated with P. fluorescens VSMKU3054 alone showed only 2 isozymes when compared to other treatments (Fig. 7).
remained higher PAL activity (34.67 μ.mol trans-cinnamic acid/min/mg Native PAGE gel electrophoresis were used to examine the expres­
protein) compared to control (Fig. 3). sion of PPO isoforms in tomato seedlings. There were difference in
expression levels of PPO isoforms among tomato seedlings treated with
3.4. Estimation of PPO P. fluorescens and challenge-inoculated with R. solanacearum, seedlings
treated with P. fluorescens alone, seedlings treated with R. solanacearum
The PPO activity was observed at higher levels in P. fluorescens alone, and control plants. In comparison to control seedlings, seedlings
treated tomato seedlings and challenged with the pathogen at 36h generated from P. fluorescens VSMKU3054 treatment and challenge-
(42.33 Δ A420 nm/min/mg protein) after pathogen inoculation inoculated with R. solanacearum (T1) exhibited four PPO isoforms:
compared to control. However, the PPO activity (37.47 Δ A420 nm/min/ PPO1, PPO2, PPO3, and PPO4 and resulted in higher levels of PPO3 and
mg protein) in R. solanacearum alone treated tomato seedlings remained PPO4 isoforms. Only two PPO isoforms were detected in the control
slightly higher than the seedlings that were treated with P. fluorescens plants and they were expressed in lower intensity levels compared to
VSMKU3054 alone (Fig. 4). other treatments (T1–T3). (Fig. 8).

5. Discussion

Rhizosphere associated microbes such as Pseudomonas spp., Strepto­

myces spp., Bacillus spp. and Trichoderma spp. significantly interacted
with plant systems by direct and indirect mechanisms for the control of
various plant diseases and remarkably support for plant growth [10,24,
42,43]. Among various rhizobacteria, Pseudomonas spp. significantly
controlled various plant diseases such as leaf blast and sheath blight of
rice, early leaf blight and leaf spot of tomato and wilt disease of tomato
[4,7,44,45] through ISR. Hence, P. fluorescens is a current approach for
the suppression of soil-borne pathogens. Our previous report has been
confirmed that P. fluorescens VSMKU3054 significantly controlled
R. solanacearum and other soil-borne fungal pathogens by the produc­
tion of various antimicrobial compounds such as 2, 4 - diacetylphlor­
oglucinol, pyoleuterin, siderophore, hydrogen cyanide, hydrolytic
enzymes and plant growth hormone IAA [2,46].
Fig. 3. Phenylalanine ammonia-lyase (PAL) activity in tomato seedlings with
In this study, we noticed that P. fluorescens VSMKU3054 induced ISR
different treatments. Control (Tomato seedlings treated with sterile distilled
water); RS - (Tomato seedlings inoculated with R. solanacearum); P. fluorescens against R. solanacearum by activating defense enzymes such as POX,
(Tomato seedlings inoculated with P. fluorescens VSMKU3054); P. fluorescens + PPO, PAL, LOX and total phenol content. Our results are concurrence
RS (Tomato seedlings treated with P. fluorescens VSMKU3054 and challenge- with the previous report [7,22] showing P. fluorescens and P. aeruginosa
inoculated with R. solanacearum. The letters above the bar represent signifi­ VMKU2 induced plant own defense mechanism like ISR for the control of
cantly different values (P = 0.05, DMRT test). wilt disease of tomato and sheath blight of rice. The present study

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P. Suresh et al. Physiological and Molecular Plant Pathology 119 (2022) 101836

Fig. 4. Polyphenol oxidase (PPO) activity in tomato

seedlings with different treatments. Control (Tomato
seedlings treated with sterile distilled water); Ral­
stonia solanacearum (RS) - (Tomato seedlings chal­
lenged with R. solanacearum); Culture (Tomato
seedlings inoculated with P. fluorescens VSMKU3054);
Culture + RS (Tomato seedlings inoculated with
P. fluorescens VSMKU3054 + R. solanacearum at
different time interval. The letters above the bar
indicate significantly different values (P = 0.05,
DMRT analysis).

Fig. 5. Lipoxygenase (LOX) activity in tomato

seedlings with different treatments. Control (Tomato
seedlings treated with sterile distilled water); RS -
(Tomato seedlings inoculated with R. solanacearum);
P. fluorescens (Tomato seedlings inoculated with
P. fluorescens VSMKU3054); P. fluorescens + RS (To­
mato seedlings treated with P. fluorescens
VSMKU3054 and challenge-inoculated with
R. solanacearum. The letters above the bar indicate
significantly different values (P = 0.05, DMRT
analysis).

Fig. 6. Total Phenolic compounds in tomato seedlings with different treatments. Control (Tomato seedlings treated with sterile distilled water); RS - (Tomato
seedlings inoculated with R. solanacearum); P. fluorescens (Tomato seedlings inoculated with P. fluorescens VSMKU3054); P. fluorescens + RS (Tomato seedlings
treated with P. fluorescens VSMKU3054 and challenge-inoculated with R. solanacearum. The letters above the bar indicate significantly different values (P = 0.05,
DMRT analysis).

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P. Suresh et al. Physiological and Molecular Plant Pathology 119 (2022) 101836

tomato seedlings treated with P. fluorescens so that the entry and colo­
nization of R. solanacearum could be avoided in tomato seedlings. PAL is
involved in the phenylpropanoid pathway which ultimately leads to the
biosynthesis of lignin. Further, lignin formation aggravates the defense
mechanism and strengthens the plant cells against infection by the
pathogen [7]. The present study revealed that higher levels of PAL ac­
tivity was observed in P. fluorescens VSMKU3054 treated tomato seed­
lings challenged with the pathogen which reached to the maximum level
at 36h after challenge inoculation. The tomato seedlings treated with
P. fluorescens VSMK3054 alone showed a moderate level of PAL activity
at 36h, but it is higher than the pathogen-treated tomato seedlings.
Vanitha and Umesha [22] have demonstrated the maximum level of PAL
activity in P. fluorescens DABBV4 pre-treated seedlings challenged with
the R. solanacearum at 12h after inoculation. Subsequently, the lower
PAL activity was found in seedlings inoculated with pathogen alone.
These results are highly concordance with our findings. Previous studies
reported that P. fluorescens Pf1 treated tomato roots challenged with the
Fusarium oxysporum f.sp. lycopersici. showed increased PAL activity at
the 4th day after pathogen inoculation [28]. The higher level of PAL was
reported in many plants such as patchouli and rice [7,47]. PAL enzyme
Fig. 7. Native PAGE analyses of peroxidase (POX) isoforms in tomato seedlings plays a major role in the production of phenolics and phytoalexins in
with various treatments. 1 - Culture (Tomato seedlings inoculated with cucumber. Polyphenol oxidases (PPO), oxidize phenolics to quinones
P. fluorescens VSMKU3054); 2 - Ralstonia solanacearum (RS) - (Tomato seedlings which are more toxic than phenolics to pathogen. The activity of PPO
challenged with R. solanacearum); 3 - Control (Tomato seedlings treated with was increased in tomato seedlings treated with P. fluorescens
sterile distilled water); 4 - Culture + RS (Tomato seedlings inoculated with VSMKU3054 and challenge-inoculated with R. solanacearum at 36 hpi
P. fluorescens VSMKU3054 + R. solanacearum). compared to control and other treatments. Konappa et al. [35] reported
that the PPO activity was significantly increased at 32 hpi in lactic acid
bacteria (LAB) treated seedlings inoculated with R. solanacearum. Xie
et al. [47] suggested that high levels of PPO activity were found in upper
leaves and lower stems at 48h after challenge inoculation with
R. solanacearum in patchouli plants.
LOX is a most important physiological enzyme implicated in plant
growth, development and induction of defense activity against plant
disease [48,49]. Furthermore, these enzymes initiate the plant defense
metabolic activity due to the biosynthesis of different antimicrobial
metabolites. The LOX is involved in the biosynthesis of jasmonic acid
and other antimicrobial oxylipins [50,51]. In our study, the LOX activity
was observed to the maximal level in pre-treated tomato seedlings
inoculated with P. fluorescens VSMKU3054 and challenged with
R. solanacearum at 48 hpi. Vanitha and Umesha [22] studied that LOX
activity was higher levels at 9 hpi in P. fluorescens DABBV4 treated
seedlings and challenged with R. solanacearum. The higher levels of LOX
were observed in tomato plants treated with Pseudomonas putida BTP1
and inoculated with Bacillus cinerea due to an increased expression of
transcripts of the two Lox isoforms namely TomLoxD and TomLoxF [19,
52]. Previous studies have shown that the seeds treated with rhizobac­
teria resulted in increased LOX activity in tomato plant leaf extracts
Fig. 8. Native PAGE analyses of polyphenol oxidase (PPO) isoforms in tomato
[53].
seedlings with various treatments. 1 - Culture + RS (Tomato seedlings inocu­
lated with P. fluorescens VSMKU3054 + R. solanacearum); 2 - Culture (Tomato
Phenolic compounds are fungitoxic in nature. Total phenol content
seedlings inoculated with P. fluorescens VSMKU3054); 3 - Ralstonia sol­ was increased in tomato seedlings treated with P. fluorescens
anacearum (RS) - (Tomato seedlings challenged with R. solanacearum); 4 - VSMKU3054 and challenge-inoculated with R. solanacearumt at 24 hpi.
Control (Tomato seedlings treated with sterile distilled water). At the same time, a moderate level of phenol content was observed in
P. fluorescens VSMKU3054 alone treated tomato seedling. However, a
revealed that all five defense enzymes have been increased in tomato very less amount of phenol content was observed in pathogen-treated
plant induction by P. fluorescens upon challenge inoculation with tomato seedlings. Our findings are in accordance with that of Anita
R. solanacearum. and Samiyappan [54], who reported that the induction of rice roots with
Peroxidase (POX) is an important enzyme for lignin biosynthesis. rhizobacteria resulted in a high amount of phenol. Anand et al. [4]
The production of POX is supportive for plant tissues and it does not stated that total phenols content was increased in Pseudomonas-treated
allow the entry of pathogens into the plants. Furthermore, it could plants followed by challenge-inoculated with A. solani and S. lycopersici.
enhance oxidative stress through the production of lipid peroxidation Hence, phenolic contents are known as potent antimicrobial metabolites
resulting in arresting of pathogen [7,26]. In the present study, higher against various pathogens.
activity of peroxidase was recorded at 24-h post-inoculation (hpi) in In our study, the native-PAGE analysis displayed four isoforms of
P. fluorescens VSMKU3054 treated tomato seedlings POX1 – POX4 at 24 hpi with R. solanacearum. The isoform POX1 showed
challenge-inoculated with R. solanacearum. prominent banding intensity in tomato seedlings treated with
The current study indicated that PAL activity was increased in P. fluorescens and challenge-inoculated with R. solanacearum. Xie et al.
(2017) reported that three POX isoforms were noticed in patchouli

6
P. Suresh et al. Physiological and Molecular Plant Pathology 119 (2022) 101836

Fig. 9. The illustration represents P. fluorescens VSMKU3054 induced ISR against R. solanacearum in tomato plants.

plants treated with R. solanacearum at different time intervals. Kavitha Acknowledgement

and Umesha [37], previously studied peroxidase isoforms in tomato
seedlings challenged with Xanthomonas axonopodis pv. vesicatoria at 15 The authors greatly acknowledged the UGC-BSR Meritorious
hpi. The native-PAGE analysis showed four isoforms of PPO at higher Fellowship, UGC-NRCBS Programme and RUSA MKU - Project (File.No.
intensity levels in P. fluorescens treated tomato seedlings compared to 011/RUSA/MKU/2021-2022), New Delhi, India for the financial sup­
control. The higher-level expression of PPO isozymes was noticed in port provided for the completion of this research work. The authors also
tomato plants treated with P. fluorescens followed by acknowledge Researchers Supporting Project No: RSP2022R465, King
challenge-inoculated with F. oxysporum f. sp. lycopersici [26]. Recently Saud University, Riyadh, Saudi Arabia for funding this research.
Arasu et al. [55] reported that bioactive potentials of plant extract on
disease causing pathopgens to tomato plant. References

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