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.