3.4 Defence enzyme activity
The expression of the four defense enzymes viz ., super oxide
dismutase (SOD), polyphenol oxidase (PPO), peroxidase (PO), and
phenylalanine ammonia lyase (PAL) were found significantly
(P<0.05 ) enhanced in the rhizobacteria (B.
pumilus. B. megaterium, B. subtilis and B. cereus ) treated
plants but not in Velum prime treatment, as compared to nematode
inoculated control. The quantified data on the enzymes evaluated shows a
gradual increase from 2 to 16 DAI in rhizobacteria pre-treated plants(Table 4 ). In nematode infected plants, pre-treated withB. pumilus , the SOD activity was 2.1, 3.2, 6.2, 8.3 & 14.5
μmol/min/mg, in B. megaterium it was 1.5, 2.8, 6.5, 7.3, & 15.6
μmol/min/mg, in B. subtilis treated plants it was 2.3, 3.5, 6.8,
8.6 & 17.5 μmol/min/mg at 0, 2, 4, 8 & 16 DAI and in B. cereustreated plants it was 1.4, 3.1, 5.5, 6 & 13.6 μmol/min/mg at 0, 2, 4, 8
& 16 DAI; respectively. In Velum prime treated plants, the SOD activity
was 0.2, 2.3, 3.0, 3.9, & 8.2 μmol/ min/mg at 0, 2, 4, 8 & 16 DAI,
respectively .The levels of these enzymes were at par in the four
rhizobacterial treatments, but were significantly enhanced wrt nematode
inoculated control and velum prime treatment. The PPO enzyme activity inB. pumilus pre-treated plants was 2.1, 2.2, 3.3, 4.1 & 6.5
μmol/min/mg, B. megaterium treated plants 1.9, 2.3, 2.9, 4.2 &
7.1 μm/ min/mg , B. subtilis treated plants it was 2.5 ,2.5, 3.8,
4.5, & 7.8 μmol/ min/mg and in B. cereus treated plants it was
2.0, 2.8, 3.9, 3.2 & 7.5 μmol/ min/mg at0, 2, 4, 8 & 16 DAI. In Velum
prime treated plants the PPO enzyme was significantly low at 0.3, 2.1,
2.7, 2.9, & 3.3 μ mol/min/mg at 0, 2, 4, 8 & 16 DAI, respectively. The
PAL enzyme activity in B. subtilis treated plants was 2.4, 2.8,
4.5, 5.5 & 8.7 μmol/min/mg, in B. pumilus pre-treated plants, it
was 2.1, 2.4, 4.1, 5.2 & 7.5 μmol/min/mg at0, 2, 4, 8 & 16 DAI, inB. megaterium pre-treated plants it was 1.8, 2.2, 3.9, 5.3 & 7.8
μm/min/mg, and in B. cereus treated plants it was 1.9, 2.0, 4.9,
4.3, & 6.8 μmol/min/mg at0, 2, 4, 8 & 16 DAI, respectively. In Velum
prime treated plants, the levels were much lower at 0.2, 1.8, 2.5, 2.6,
& 3.6μ m/min/mg at0, 2, 4, 8 & 16 DAI, respectively as compared to
nematode inoculated control. The PO enzyme activity shown in B.
pumilus treated plants was 2.3, 2.2, 3.6, 4.8 & 8.5 μmol/ min/mg, inB. megaterium treated plants it was 1.8, 2.4, 3.8, 4.7 & 9.5
μmol/ min/mg, in B. subtilis treated plants, it was 2.6, 2.5,4.6,
5.3 & 10.2 μmol/min/mg and in B. cereus treated plants shown it
was 2.8, 2.0, 3.6, 4.5 & 8.5 μm/ min/mg at0, 2, 4, 8 & 16 DAI. In
Velum prime treated plants, the levels of PO were at a low of 0.3, 2.4,
2.6, 3.0 & 3.7 μmol/ min/mg at0, 2, 4, 8 & 16 DAI, respectively as
compared to control. Among the above defense enzymes the activity of SOD
was found highest followed by PAL, PO and PPO. In all the rhizobacterial
treatments, a gradual increase in enzyme levels were observed with an
increase in time period from 2 to 16 DAI, as compared to control. Thus
application of all the four rhizobacteria increased the levels of
defense enzymes in the nematode free plants which further increase upon
nematode inoculation. The levels of all four enzymes were at par on
respective 5 days of observation and were significantly higher than that
observed in velum prime treated plants. The rhizobacteria thus up
regulated the defense mechanism in plants, unlike the chemical
nematicide Velum Prime.
DISCUSSION
The rhizosphere harbours many species of bacteria, many of which are
known to induce resistance in plants against soil borne pests and
pathogens. Indirect antagonism of PGPR against PPN that occurs by ISR,
also referred to as PGPR-mediated priming, systematically equips the
plants to cope with environmental constraints, actuating faster and/or
stronger defense responses (adaption) to a subsequent exposure to
various biotic and abiotic stresses. ISR is nonspecific in nature, and
provides plants defense responses against various pests and pathogens.
The rhizosphere bacterial isolates like Bacillus pumilus,
Paenibacillus castaneae, Pseudomonas fluorescens, Bacillus subtilis,
Bacillus cereus , Arthrobotrys oligospora, Bacillus megaterium,
Pseudomonas striata and Paenibacillus polymyxa directly and
indirectly suppress nematode infestation and promote plant growth
(Subedi et al ., 2020). ISR induced by PGPR typically employs
jasmonic acid (JA) and ethylene (ET) hormone signaling (Ahmed et
al ., 2022). In the present investigation, the rhizobacteria elicited
enhancement in defense gene expression and subsequently the enzymes that
confer resistant reactions in tomato against M. incognitainfection. The qRT-PCR results exhibited that the expressions of
resistance genes viz., PR1-1b, CAT, and JERF3 in tomato plants were
different, in the four rhizobacteria (B. pumilus. B. megaterium,
B. subtilis and B. cereus ) treatments at intervals of 0, 2, 4, 8
& 16 days DAI as compared with untreated control (Fig. 1) .
Among the three genes, the maximum upregulation was observed in PR-1b,
followed by CAT and JERF3. PR1-1b gene has been referred as an immune
marker and the target gene for salicylic acid (Spoel & Dong, 2012; Fu
& Dong, 2013).The hypersensitive response of PR-1b has been reported to
be induced by activation of Jasmonic acid (JA) signalling pathway and
ethylene (ET) in tomato (Mollinari and Leonatti, 2019). Furthermore, the
rhizobacterial treated plants expressed JERF3 gene at 2- 6 folds and CAT
gene at 3.5-6.0 folds, compared to untreated control. JERF3 and CAT
genes encode for ERF proteins, a trans-acting factor responding to both
ET and JA. Besides, the expression of CAT gene encodes for catalase,
which neutralizes the toxic hydrogen peroxide produced in plant defense
against pathogens and parasites. The defense gene expression during
rhizobacterial-root interactions showed an initial overexpression toM. incognita infection followed by a defense repression. The
process shares similarities with root mycorrhized plants where
mycorrhizia induced resistance (MIR) (Pozo et al ., 2007).
Comparably, our bacterial treated plants were primed for activation of
defensive genes against M. incognita infection in tomato. The
primed plants respond faster to biotic attacks and make stronger defense
activation (Molinari S, Leonetti ,2019). The activities of defense
enzymes viz ., SOD, PPO, PO, and PAL were found significantly
(P<0.05 ) expressed in rhizobacteria (B. pumilus.
B. megaterium, B. subtilis and B. cereus ) treated tomato plants
infected with M. incognita as compared to untreated plants. The
activity of the above enzymes showed a gradual increased from 2 to 16
days after nematode inoculation in rhizobacteria treated roots. Among
the enzymes, the activity of SOD was found highest followed by PAL, PO
and PPO. Nematode infestation leads to the production of superoxide
anions which are highly reactive. SOD is a constitutive enzyme produced
by aerobic organisms in response to biotic stresses. The enzyme
scavenges reactive oxygen species (ROS) by producing hydrogen peroxide
and alongwith catalase maintains the cellular levels of ROS below the
threshold levels that trigger cell death due to necrosis and
hypersensitive reaction leading to reduced nematode infection.
Peroxidases (PO) catalyze the generation of ROS. PPO can inhibit the
pest by producing quinones, reduce bioavailability of proteins and
nutrients for the pest/pathogen, create lignin-like physical barriers
and participate in the production of ROS. Similar upregulation of
defense enzymes by rhizobacteria treated tomato against root knot
nematode infection has been reported earlier (Ramazan et al .,
2018; Sharma and Sharma, 2016). The nematicide (Velum prime) treated
plants did not show any enhancement in gene expression or enzyme
activities as compared to untreated control.
The bioefficacy trials in pots revealed significant higher reduction in
root galling (81-91%), average number of egg masses per root (16-43 %)
and reproduction factor (51-67%) in rhizobacterial treatments as
compared to control (Fig 2-4 ). Besides, the four rhizobacterial
treatments also resulted in a significant enhancement in the
photosynthetic and transpiration rate, though no significant difference
was found with respect to stomatal conductance and leaf temperature, as
compared to untreated control. Thus the rhizobacteria are environment
friendly substitutes to agrochemicals for pest management. Among
rhizobacteria, the Bacillus spp. are considered to be good
options because they can quickly replicate and colonize plants, tolerate
harsher environments, and easily form endospores. They are documented to
affect a broad spectrum of plant pathogens including PPN and can promote
plant growth and help the plant adapt abiotic stresses, enhancing yield
potential (Ahmed at al. 2022).
The present study revealed that the pretreatment of tomato plants with
nematicidal rhizobacterial isolates resulted in the activation of
defense response by enhancing the expression of genes and enzymes
governing resistance, reducing the nematode infection as indicated by
gall index and reproduction factor of the nematode. The photosynthetic
and transpiration rate of plants also increased, as compared to
untreated control. Therefore, the tested rhizobacterial isolates are a
promising approach for management of M. incognita and for
improving the growth and productivity of plants.
ACKNOWLEDGMENTS This study was supported by fellowship granted
by Post Graduate School, ICAR-IARI, Pusa for conducting PhD. Research.