Native Trichoderma Induced the Defense-Related Enzymes and Genes in Rice against Xanthomonas oryzae pv. oryzae (Xoo)

The application of Trichoderma is a form of biological control that has been effective in combating Xanthomonas oryzae pv. oryzae, the causative agent of the devastating disease known as bacterial blight of rice. In this present study, four strains of Trichoderma, viz., T. paraviridescens (BDISOF67), T. erinaceum (BDISOF91), T. asperellum (BDISOF08), and T. asperellum (BDISOF09), were collected from the rice rhizosphere and used to test their potentiality in reducing bacterial blight. The expression patterns of several core defense-related enzymes and genes related to SA and JA pathways were studied to explore the mechanism of induced resistance by those Trichoderma strains. The results primarily indicated that all Trichoderma were significantly efficient in reducing the lesion length of the leaf over rice check variety (IR24) through enhancing the expression of core defense-related enzymes, such as PAL, PPO, CAT, and POD activities by 4.27, 1.77, 3.53, and 1.57-fold, respectively, over control. Moreover, the results of qRT-PCR exhibited an upregulation of genes OsPR1, OsPR10, OsWRKY45, OsWRKY62, OsWRKY71, OsHI-LOX, and OsACS2 after 24 h of inoculation with all tested Trichoderma strains. However, in the case of RT-PCR, no major changes in OsPR1 and OsPR10 expression were observed in plants treated with different Trichoderma strains during different courses of time. Collectively, Trichoderma induced resistance in rice against X. oryzae pv. oryzae by triggering these core defense-related enzymes and genes associated with SA and JA pathways.


Introduction
Rice (Oryzae sativa L.) is one of the major cereal crops belonging to the Poaceae family, especially in Asian countries including China, India, Bangladesh, Thailand, and Vietnam [1,2]. With a constantly rising production of 3.6 crore tons of rice, Bangladesh stands in the third position globally in rice production after China and India [3]. However, bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is among the thirty-two rice diseases reported in Bangladesh, which can cause 5.8-30.4% yield loss depending on the crop stage and environmental factors [4,5]. For systemic bacterial blight infection, X. oryzae pv. oryzae enters the rice leaf generally by wounds or water pores of wavy leaf edges, multiplies in the intercellular spaces of the underlying epithelial tissues, and then moves to the xylem vessels [6]."Within a few days bacterial cells and EPS fill the xylem vessels and ooze out from hydathodes, forming beads or strands of exudate on the leaf surface, a characteristic sign of the disease and a source of secondary inoculum" [7].
Chemical management, biological control, and genetic resistance have all been used to manage bacterial leaf blight disease in rice plants. Despite the fact that the rising population encourages the use of more chemical inputs, such management strategies are A total of 200 fungal isolates were isolated and purified from the rice rhizosphere. Out of these, four fungal isolates, viz., BDISOF67R, BDISOF91R, BDISOF08R, and BDISOF09R, were identified as antagonists against X. oryzae pv. oryzae ( Figure 1A) that inhibited the growth of X. oryzae pv. oryzae by 67.51%, 52.19%, 24.06%, and 23.33%, respectively, over control ( Figure 1B). evaluate the potentiality of Trichoderma isolates in controlling BB of rice. Moreover, we assessed the expression patterns of several core defense-related enzymes and genes which are related to SA and JA pathways to explore the mechanism of induced systemic resistance by Trichoderma strains in controlling X. oryzae pv. oryzae.

Figure 1. (A)
In vitro growth inhibition of X. oryzae pv. oryzae by antagonistic fungal isolates (BDISOF67R: obtained from rice rhizosphere from Bogura; BDISOF91R: obtained from rice rhizosphere from Hobigonj; BDISOF08R and BDISOF09R: both obtained from rice rhizosphere from Jashore) (Photographs taken after 72 h of inoculation). (B). Error bar chart of percent growth inhibition of X. oryzae pv. oryzae by antagonistic fungal isolates. (* represents significance at 5% level of significance. Similar letters at the top of bars indicate the treatment means are statistically similar).

Molecular Identification of Fungal Isolates BDISOF67R, BDISOF91R, BDISOF08R, and BDISOF09R
Fungal isolates BDISOF67R, BDISOF91R, BDISOF08R, and BDISOF09R were identified by PCR amplification of the internal transcribed spacer (ITS) region using ITS-1 and ITS-4 primers and PCR products were then sequenced. The results of PCR showed an amplification size of 600 bp and confirmed that the antagonist organisms are fungi ( Figure  2).

Figure 1. (A)
In vitro growth inhibition of X. oryzae pv. oryzae by antagonistic fungal isolates (BDISOF67R: obtained from rice rhizosphere from Bogura; BDISOF91R: obtained from rice rhizosphere from Hobigonj; BDISOF08R and BDISOF09R: both obtained from rice rhizosphere from Jashore) (Photographs taken after 72 h of inoculation). (B). Error bar chart of percent growth inhibition of X. oryzae pv. oryzae by antagonistic fungal isolates. (* represents significance at 5% level of significance. Similar letters at the top of bars indicate the treatment means are statistically similar).

Molecular Identification of Fungal Isolates BDISOF67R, BDISOF91R, BDISOF08R, and BDISOF09R
Fungal isolates BDISOF67R, BDISOF91R, BDISOF08R, and BDISOF09R were identified by PCR amplification of the internal transcribed spacer (ITS) region using ITS-1 and ITS-4 primers and PCR products were then sequenced. The results of PCR showed an amplification size of 600 bp and confirmed that the antagonist organisms are fungi ( Figure 2).

Effect of Some Selected Antagonistic Trichoderma Isolates on the Reduction of Lesion Length in Susceptible Check Cultivar (IR24) Caused by X. oryzae pv. oryzae
The results of plant inoculation (rice seeds first treated with Trichoderma formulations and then foliar application of Trichoderma spp.) showed a significant reduction of lesion length in plants sprayed with formulated Trichoderma spp. as compared with untreated control (Figure 3; Table 2).    On the other hand, the maximum (647 µmol transcinnamic acid min −1 g −1 protein) PAL activity was recorded for plants treated with BDISOF91 (T. erinaceum) at 72 HAI, while the minimum (555 µmol transcinnamic acid min −1 g −1 protein) PAL activity was at 48 HAI, which showed 4.05 and 2.32 times more PAL activity over control, respectively. However, plants treated with BDISOF08 (T. asperellum) showed the maximum (706 µmol transcinnamic acid min −1 g −1 protein) PAL activity at 24 HAI, while the minimum (530 µmol transcinnamic acid min −1 g −1 protein) PAL activity was recorded at 72 HAI, which were 2.85 and 3.32 times higher over control, respectively. Lastly, plants treated with BDISOF09 (T. asperellum) accounted for the maximum (682 µmol transcinnamic acid min −1 g −1 protein) PAL activity at 72 HAI, while the minimum (556 µmol transcinnamic acid min −1 g −1 protein) PAL activity was recorded at 24 HAI, which demonstrated 4.27 and 2.25 times higher PAL activity over control, respectively.

Influence of Selected Trichoderma Isolates in Induction of Catalase (CAT) Activity in
Response to X. oryzae pv. oryzae From Table 4, plants treated with BDISOF67 (T. paraviridescens) showed the maximum (273 µmol H 2 O 2 min −1 g −1 ) CAT activity at 24 HAI, while the minimum (162 µmol H 2 O 2 min −1 g −1 ) CAT activity was recorded at 72 HAI, which were 5.46 and 1.34 times higher over control, respectively. On the other hand, the maximum (264 µmol H 2 O 2 min −1 g −1 ) CAT activity was recorded for plants treated with BDISOF91 (T. erinaceum) at 24 HAI, while the minimum (118 µmol H 2 O 2 min −1 g − 1) CAT activity was at 48 HAI, which showed 5.28 and 0.98 times more CAT activity over control, respectively. However, plants treated with BDISOF08 (T. asperellum) showed the maximum (201 µmol H 2 O 2 min −1 g −1 ) CAT activity at 144 HAI, while the minimum (59 µmol H 2 O 2 min −1 g −1 ) CAT activity was recorded at 72 HAI, which were 1.94 and 0.49 times higher over control, respectively. Lastly, plants treated with BDISOF09 (T. asperellum) accounted for the maximum (367 µmol H 2 O 2 min −1 g −1 ) CAT activity at 144 HAI, while the minimum (78 µmol H 2 O 2 min −1 g −1 ) CAT activity was recorded at 72 HAI, which demonstrated 3.53 and 0.64 times higher CAT activity over control, respectively. As is illustrated by Table 5, plants treated with BDISOF67 (T. paraviridescens) showed the maximum (454 units g −1 min −1 FW) PPO activity at 24 HAI, while the minimum (247 units g −1 min −1 FW) PPO activity was recorded at 72 HAI, which were 1.77 and 1.55 times higher over control, respectively. On the other hand, the maximum (434 units g −1 min −1 FW) PPO activity was recorded for plants treated with BDISOF91 (T. erinaceum) at 24 HAI, while the minimum (271 units g −1 min −1 FW) PPO activity was at 72 HAI, which showed 0.95 and 1.7 times more PPO activity over control, respectively. However, plants treated with BDISOF08 (T. asperellum) showed the maximum (428 units g −1 min −1 FW) PPO activity at 24 HAI, while the minimum (222 units g −1 min −1 FW) PPO activity was recorded at 144 HAI, which were 0.98 and 0.82 times higher over control, respectively. Lastly, plants treated with BDISOF09 (T. asperellum) accounted for the maximum (403 units g −1 min −1 FW) PPO activity at 24'HAI, while the minimum (284 units g −1 min −1 FW) PPO activity was recorded at 48 HAI, which demonstrated 0.94 and 1.19 times higher PPO activity over control, respectively.

Influence of Selected Trichoderma
Isolates in Induction of Peroxidase (POD) Activity in Response to X. oryzae pv. oryzae Table 6 indicates that plants treated with BDISOF67 (T. paraviridescens) showed the maximum (1751 units min −1 g −1 FW) POD activity at 144 HAI, while the minimum (850 units min −1 g −1 FW) POD activity was recorded at 24 HAI, which were 1.47 and 1.12 times higher over control, respectively.

Discussion
Rhizosphere microorganisms that are beneficial to plants can greatly enhance their resistance to pathogens by inducing the production of defense genes and proteins [11,15,39,40,57]. Trichoderma isolates in plants have been shown to protect plants from biotic stressors by, among other mechanisms, inducing systemic resistance, programmed cell death, triggering signaling cascades, callose deposition, induction of phytoalexins and other secondary metabolites, and producing antibacterial reactive oxygen species [13,[58][59][60]. The findings of this study show that when infected with X. oryzae pv. oryzae, rice OsACS2 involved in SA and JA acid pathways by q-PCR in rice treated with Trichoderma in response to X. oryzae pv. oryzae over untreated control. Untreated control: seed treatment with only distilled water (T. paraviridescens (BDISOF67), T. erinaceum (BDISOF91), T. asperellum (BDISOF08), and T. asperellum (BDISOF09), respectively). * represents significance at 5% level of significance. Similar letters at the top of bars indicate the treatment means are statistically similar.

Discussion
Rhizosphere microorganisms that are beneficial to plants can greatly enhance their resistance to pathogens by inducing the production of defense genes and proteins [11,15,39,40,57]. Trichoderma isolates in plants have been shown to protect plants from biotic stressors by, among other mechanisms, inducing systemic resistance, programmed cell death, triggering signaling cascades, callose deposition, induction of phytoalexins and other secondary metabolites, and producing antibacterial reactive oxygen species [13,[58][59][60]. The findings of this study show that when infected with X. oryzae pv. oryzae, rice plants primed with beneficial Trichoderma strains had higher activity of defense-related enzymes and genes related to SA and JA pathways in leaves compared to control plants.
In the current study, the lesion length of rice leaves was reduced, ranging from 31.25% (BDISOF09: T. asperellum) to 87.5% (BDISOF08: T. asperellum) in plants treated with Trichoderma isolates. This significant reduction in lesion development in the in vitro pathogenicity assay signifies that the rice plants primed with Trichoderma successfully protected against the development of bacterial blight disease. The findings of this study correlate with the reports from Jambhulkar et al., Gangwar (2013), and Manmeet and Thind, who reported that Trichoderma species were found to reduce the lesion length as well as the severity of bacterial blight diseases in rice plants [18,19,61]. However, Divya et al. revealed that a T. asperellum isolate was 17.20% more effective in controlling lesion length development than T. atroviride [57]. Meanwhile, T. harzianum was found to be the best in managing bacterial blight, giving 52.66% and 26.66% reduction in disease severity and incidence, respectively [62]. In another study, Kariuki et al. found that Trichoderma isolate T1 was found to be the most effective in reducing bacterial wilt incidence by more than 61.66% compared to the control [63]. Interestingly, T. paraviridescens (BDISOF67) and T. erinaceum (BDISOF91) were able to significantly reduce the radial growth of X. oryzae pv. oryzae before coming in direct contact with Trichoderma strains. This might be because of the fact that Trichoderma strains release antimicrobial compounds in the medium, which in turn reduces or stops the multiplication rate of X. oryzae pv. oryzae in vitro [11,[64][65][66]. A similar report by Gangwar and Sinha demonstrated that Trichoderma spp. was able to inhibit the radial growth of X. oryzae pv. oryzae at a maximum of 100% [13]. As stated by Kannan et al., among different isolates of Trichoderma, TAIK 1 was found to significantly inhibit more than 40% growth of X. oryzae pv. oryzae in vitro [8]. A similar trend was observed by Rubio et al., who revealed that T. parareesei showed antagonistic action against Pythium irregulare due to its production of cellulolytic enzymes, which break down the cellulose-based cell walls of the fungus [67].
A key discovery of this research is the mechanism by which Trichoderma strains reduce the bacterial blight of rice by inducing resistance against X. oryzae pv. oryzae. We noticed an enhanced activity of defense-related enzymes, viz. PAL, CAT, PPO, and POD, in rice on seed priming with Trichoderma strains. Upregulation of these enzymes leads to the production of signaling molecules, like SA and JA, and metabolites with defense functions, including phenols, phytoalexins, lignin, as well as flavonoids, and antimicrobial activities in plants [34,59,68,69]. Our results show PAL activity was upregulated a maximum of four times over control at 72 HAI when plants were treated with T. erinaceum and T. asperellum. These findings, on the whole, are in agreement with the findings of [39,[70][71][72]. Enhanced activity of defense-related enzymes was also reported by Adss et al., who showed that POD, PPO, and PAL activity were significantly increased over control in all treatments after treating A. solani with T. harzianum [37]. Similar outcomes were observed while inducing the activity of PPO and POD in chickpeas [22] and rice [40] treated with T. harzianum and Bacillus spp., respectively, against Sclerotium rolfsii and Pyricularia oryzae infection. Mei et al. discovered that cucumbers treated with Trichoderma strains against Fusarium wilt had a higher CAT expression level [73]. In contrast, plants inoculated with T. asperellum for this study expressed elevated levels of catalase enzyme in leaves at 24 HAI over control, which coincides with Samal et al., who reported that biopriming with T. erinaceum resulted in reduced bacterial blight by inducing CAT activity in rice against X. oryzae pv. oryzae [38]. These findings clearly support that PAL, CAT, PPO, and POD activity might be significant enzymes for the induction of disease resistance to X. oryzae pv. oryzae in rice plants. The next step of our research is to investigate the expression of other defense-related enzymes in rice plants treated with Trichoderma strains.
RT-PCR was employed in this study to evaluate the expression patterns of several defense-related genes (OsPR1, OsPR10, OsWRKY45, OsWRKY62, OsWRKY71, OsHI-LOX, and OsACS2) that were reported to be induced by niclosamide, a chemical inducer in rice plants treated with different Trichoderma strains [41]. Our results clearly indicate that the expression of the genes, viz., OsWRKY45, OsWRKY62, OsWRKY71, OsHI-LOX, and OsACS2, were significantly higher at 144 HAI after X. oryzae pv. oryzae infection, in contrast to other time course expressions, suggesting that they might be responsible for the enhanced resistance to bacterial blight in rice plants. No major changes in OsPR1 and OsPR10 expression were observed in plants treated with different Trichoderma strains, indicating that these genes might not be primarily responsible for the enhanced resistance of rice plants to bacterial blight. These results are consistent with those reported by [41,46,49,67,74]. Meanwhile, Contreras-Cornejo et al. revealed that the expression of Lox2 A genes in response to T. virens or T. atroviride indicated the involvement of SA and/or JA pathways in the defense signaling pathway activated by those fungi [49]. However, our analysis revealed an antagonistic relationship between the genes of the SA and JA synthesis pathways, in which the presence of OsHI-LOX and OsACS2 in the JA assay suppressed the expression of OsPR1 and OsPR10 in the SA assay. This strategy is backed up by Mur et al., who reported that SA and JA act antagonistically when used in higher concentrations [75]. In contrast, De Vleesschauwer et al. developed a model that shows how SA and JA pathways might combine to form a shared defense mechanism that is effective against various sorts of invaders [76].
Confirmation through the endpoint of qRT-PCR complements the expression of defense-related genes involved in SA and JA acid pathways as the maximum expression after 24 h of inoculation with all tested Trichoderma strains. This strategy is also supported by Ding et al., who showed that PR-1.1 was induced in response to treatments with SA or methyl jasmonate (MeJA), while PR3 and LOX2 responded positively to MeJA treatment after 24 h [47]. In addition, overexpression of LOX genes has been demonstrated to be necessary for giving resistance to diseases caused by bacteria [44,45,77]. In another study, Uji et al. stated that the Trichoderma strain induced the expression of Lox1 genes against the rice bacterial blight pathogen X. oryzae pv. oryzae [78]. In our study, higher levels of expression of the OsWRKY45, OsWRKY62, and OsWRKY71 genes were observed in plants raised from treated seeds and sprayed with different Trichoderma strains. These findings are totally in agreement with Kim et al., who demonstrated that niclosamide blocks the development of rice leaf blight by inducing the expression of defense-related genes, including OsPR1, OsPR10, OsWRKY45, OsWRKY62, OsWRKY71, OsHI-LOX, and OsACS2 [38]. However, some studies show that multiple members of the WRKY gene family involved in JA response pathways, such as WRKY, bZIP [20], OsWRKY80, OsWRKY4 [48], OsWRKY9, OsWRKY45, OsWRKY5, OsWRKY28, OsWRKY29 [79], WRKY18, WRKY40 [80], and Os-WRKY45 [81] activated the defense response in the interaction with the beneficial fungus Trichoderma species on susceptible rice plants. Together with the findings, we support that rice plants treated with beneficial Trichoderma species can significantly induce resistance against X. oryzae pv. oryzae by the up-regulation of defense-related enzymes and marker genes of the SA and JA pathways. Conversely, it would be interesting for our study to test the SA and JA levels in Trichoderma-treated rice plants.

Identification of Trichoderma Species Antagonistic to X. oryzae pv. oryzae
To identify the Trichoderma species antagonistic to X. oryzae pv. oryzae, rice plant samples with root systems were collected from 40 rice growing districts, representing 30 agro-ecological zones of Bangladesh. The Trichoderma species were then isolated following the dilution plate technique (up to 10 −3 concentration) on PDA (Potato Dextrose Agar; Difco™) plates at a temperature of 28 • C. To assess the antagonistic activity of Trichoderma species against X. oryzae pv. oryzae, in vitro growth assay was performed as described by Tian et al. [82] with slight modifications. Briefly, X. oryzae pv. oryzae inocula were prepared from 48 h old cultures and a 100 µL bacterial cell suspension (1 × 10 8 cells/ml) was spread on NBY (Nutrient Broth Yeast) plates. A 5 mm mycelial block of Trichoderma species was then placed at the centre of the plate and incubated at 28 • C for 3 days. The growth inhibition of X. oryzae pv. oryzae by the isolates of Trichoderma as indicated by clear halo zones were then recorded and expressed in percentage over control using the following formula: % Growth Inhibition = Diameter of fungal colony with clear halo zone − Diameter of the fungal colony Diameter of fungal colony with clear halo zone The genomic DNA of these Trichoderma isolates was then extracted by using Wizard ® Genomic DNA Purification Kit solution (Promega, Madison, WI, USA) according to the manufacturer's instructions. PCR reactions were performed with the universal primer sets ITS 1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS 4 (5 TCCTCCGCTTATTGATATGC-3 ) according to White et al. [83]. The PCR amplification was performed in a T100 thermal cycler (BioRed) using the initial denaturation at 95 • C for 5 min, denaturation at 95 • C for 30 s, annealing at 55 • C for 1 min, extension at 72 • C for 1 min for 35 cycles and final extension at 72 • C for 6 min. The molecular size of the resulting PCR product was analyzed on a 1.0% agarose gel. PCR-amplified internal transcribed spacer (ITS) regions were then subjected to partial nucleotide sequencing using forward primer ITS1 in the Macrogen Lab, South Korea, via Biotech Concern Bangladesh. The DNA sequences were then compared using the BLAST (Basic Local Alignment Search Tool) program to identify their closest relatives.

Formulation of Trichoderma Species, Seed Priming, and Foliar Spray of Formulated Trichoderma Species
A mycelial disc (5 mm diameter) of each Trichoderma isolate was inoculated in 100 mL PDB broth. The number of colony-forming units was counted after 7 days. The mycelial mat along with conidia from potato dextrose broth (PDB) was mixed thoroughly with autoclaved talcum powder pretreated with 0.5% carboxy methyl cellulose (CMC) (5 g CMC dissolved in 100 mL water mixed with 1 kg talcum powder). The mixture was air-dried in a laminar flow hood and kept in plastic bags at 30% moisture content. The formulated fungal antagonists were then kept at 4-8 • C in the refrigerator. The rice seeds of IR24 were then treated with Trichoderma formulations at 10 g per kg rice seeds for half an hour. Treated seeds were sown in the earthen pots, and then 30 days old seedlings were transplanted in the pot following a completely randomized design (CRD) with 3 replications. The Trichoderma formulations were then sprayed at 40, 50, 65, and 75 days after transplanting (DAT). Formulated fungal powders were suspended in water to prepare the fungal solution at 0.5%, i.e., 5 g/L water and sprayed on the plant surface with the help of a sprayer.

Inoculation of Rice Plants with X. oryzae pv. oryzae
The X. oryzae pv. oryzae was cultured in NBY agar medium at 28 • C for 48 h and then resuspended in sterile distilled water at a cell density of 5 × 10 8 cells/ml measured by Spectrophotometer. The leaves of each hill in each replication were inoculated by the clip-inoculation method of Kauffman [84]. Artificial inoculation of X. oryzae pv. oryzae was done at 60 DAT. After inoculating, the pots were transferred to an artificial growth chamber maintaining temperature (28 • C) and relative humidity (90%). Data was collected on lesion length at 7, 14, and 21 days after inoculation (DAI). The best or potential Trichoderma antagonistic to X. oryzae pv. oryzae was identified by the data analysis.

Expression Analysis of Some Defense-Related Enzymes in Rice in
Response to X. oryzae pv. oryzae Ten rice leaf samples for each treatment were collected at 24, 48, 72, and 144 h after inoculation (HAI) in zipper bags. The collected leaf samples were frozen with liquid nitrogen and ground into powder for either RNA extraction immediately, or the ground samples were stored at −80 • C for future use. Then, to explore the mechanism of Trichoderma mediated induced resistance in rice against X. oryzae pv. oryzae, the activities of core defenserelated enzymes, viz., PAL, CAT, PPO, and POD, were assessed.

Phenylalanine Ammonia-Lyase (PAL)
The PAL activity was analyzed at 290 nm wavelength by measuring the conversion of L-phenylalanine into ammonia and trans-cinnamic acid [85]. For this, 1 g rice leaves was homogenized in 5 mL of 0.1 M borate buffer, pH 7.0, containing 0.1 g polyvinyl polypyrrolindone (PVP) at 4 • C with a pestle and mortar. The homogenate was centrifuged at 10,000× g for 30 min at 4 • C. The supernatant was collected and used in the enzyme assay. The reaction mixture included 0.4 mL of enzyme extract, 0.5 mL of 0.1 M borate buffer (pH 8.8), and 0.5 mL of 12 nm L-phenylalanine in the same buffer. The reaction mixture was incubated at 30 • C for 30 min in a water bath. The reference cell was 0.4 mL of enzyme extract and 1.0 mL of borate buffer. The amount of trans-cinnamic acid synthesized was calculated using its absorption coefficient of 9630 µmol s −1 g −1 .

Catalase (CAT)
To study the CAT activity, the decreased amount of hydrogen peroxide (H 2 O 2 ) was determined at 240 nm wavelength [86]. "The CAT assay mixture of 3 mL consisted of 0.05 mL extract, 1.5 mL phosphate buffer (100 mM buffer, pH 7.0), 0.5 mL H 2 O 2 , and 0.95 ml distilled water. A decrease in the absorbance was recorded at 240 nm. The CAT activity was expressed as µmol of H 2 O 2 oxidized per minute per gram FW".

Polyphenol Oxidase (PPO)
The PPO was determined at 280 nm wavelength using L-tyrosine as the substrate [87]. For this, 0.5 mL of leaf extracts were used for the PPO (catechol oxidase) assay. It was mixed with 250 µL of 50 mM sodium phosphate buffer. The rate of increase in absorbance at 525 nm was measured for 2 min after the addition of 500 µL 0.1 M catechol. The OD 525 was measured using a T80 UV/VIS Spectrophotometer (pg instruments). There were 3 replications for each sample. One unit of the enzyme was defined as the amount of enzyme that increases the OD 525 value by 0.01.

Peroxidase (POD)
The POD was assayed using guaiacol as a substrate at 470 nm wavelength and expressed as changes in absorbance min −1 g −1 fresh wt of tissue [88]. Rice leaves (1 g) were homogenized in 5 mL 0.1M phosphate buffer (pH 7.0) at 4 • C. Then it was centrifuged at 10,000× g for 20 min at 4 • C and supernatant was collected. In a sample cuvette of the spectrophotometer, 1.5 mL of 0.05M pyrogallol and 0.1 mL of enzyme extract were added. The absorbance reading will be adjusted to zero at 420 nm in the T80 UV/VIS Spectrophotometer (pg instruments). To initiate the reaction, 0.1 mL of 1% H 2 O 2 was added to the sample cuvette. The changes in absorbance were recorded at 30 s intervals.

Assessment of Differential Expression in Plants Treated with Trichoderma
To study the Salicylic acid (SA) and Jasmonic acid (JA) pathway-mediated induced resistance in rice by Trichoderma, a susceptible check variety (IR24) was used. Seven genes were selected for their induced expression by Trichoderma which were shown to be induced resistance in rice against BB.

RNA Extraction and cDNA Synthesis
Total RNA was extracted from 30-50 mg of ground rice leaf tissues using the kit SB total RNA isolation system (Promega, Madison, WI, USA) according to the manufacturer's instructions. First-strand complementary DNA was then synthesized using 5 µg of total RNA using Go-script reverse transcriptase (Promega, Madison, WI, USA) and primers [Oligo(dT) 15 (0.5 µg/reaction) and/or Random Primer (0.5 µg/reaction) or gene-specific primer (10-20 pmol/reaction)]. The following procedures were carried out: primer annealing at 25 • C for 5 min, DNA polymerization at 42 • C for up to 1 h, and deactivation of reverse transcriptase at 70 • C for 15 min.

Primer and Reverse Transcription (RT)-PCR
RT-PCR was performed using Hot-start Go Taq master mix (Promega, Madison, WI, USA) following the instruction of the kit's manual. The following primers (Table 7) were used for the analyses of the expression of some selected marker genes of SA and JA pathways. Table 7. List of primers for the expression study of some selected defense-related genes of rice against X. oryzae pv. oryzae.

Genes
Primer Name Sequence ( The PCR amplification reaction was performed under the following thermal cycling conditions: 95 • C for 10 min, 45 cycles of 95 • C for 10 s, 60 • C for 10 s, and 72 • C for 10 s. The expression level of different defense-related genes was compared based on the intensity of the band as compared with the untreated control. The 18S rRNA gene was used as the internal control. The following primers were used (Table 7).

Real-Time qPCR Assay
The q-PCR assay was performed using Luna q-PCR universal master mix (New England Biolab, London, UK). PCR reaction mix contained Luna universal q-PCR (4.5 µL), forward primer 0.25 µL (10 µm), reverse primer 0.25 µL (10 µm), template cDNA 1 µL, and nuclease-free water 4.0 µL were used for quantification of relative expression. The PCR conditions were as follows: initial denaturation was done at 95 • C for 60 s in one cycle. The denaturation was done at 95 • C for 15 s, extension at 60 • C for 30 s in 40-45 cycles, annealing at 60 • C for 10 s, and melt curve for 60-95 • C for various seconds in one cycle. The relative expression was calculated using the following formula: Relative expression value = 2 −∆∆CT [89]. CT value of the target gene was normalized using the CT value of 18S rDNA.

Statistical Analysis
The data were subjected to analysis of variance, and Duncan's multiple range test was used to differentiate means at 5%. The error bars in all figures indicate the standard error of the mean. For data analysis, Minitab (version 17) software was used.

Conclusions
Our study offers a glimpse into the molecular mechanism of Xoo-rice interactions with Trichoderma isolates, and reveals the upregulated expression of the selected defense related enzymes and genes related to the SA and JA pathways. Based on the key findings, Trichoderma mediated induced resistance in rice against Xoo was summarized using the following proposed model ( Figure 6).

Conclusions
Our study offers a glimpse into the molecular mechanism of Xoo-rice interactions with Trichoderma isolates, and reveals the upregulated expression of the selected defense related enzymes and genes related to the SA and JA pathways. Based on the key findings, Trichoderma mediated induced resistance in rice against Xoo was summarized using the following proposed model ( Figure 6). In this proposed model ( Figure 6), Trichoderma triggers the SA and JA pathways and enhances the expression of some SA and JA responsive genes, and thus induced resistance in rice against Xoo. All four Trichoderma strains used in this study also enhance the expression of WRKY factors which might be either SA/JA-dependent or independent ( Figure 6). However, expression levels of PR1 and PR10 genes primarily indicated a weak antagonism of SA and JA pathways in rice against Xoo ( Figure 6). Therefore, an RNA-seq study is required to understand the underlying mechanisms by which these Trichoderma strains promote induced system resistance ( Figure 6).

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1. In this proposed model (Figure 6), Trichoderma triggers the SA and JA pathways and enhances the expression of some SA and JA responsive genes, and thus induced resistance in rice against Xoo. All four Trichoderma strains used in this study also enhance the expression of WRKY factors which might be either SA/JA-dependent or independent ( Figure 6). However, expression levels of PR1 and PR10 genes primarily indicated a weak antagonism of SA and JA pathways in rice against Xoo ( Figure 6). Therefore, an RNA-seq study is required to understand the underlying mechanisms by which these Trichoderma strains promote induced system resistance ( Figure 6).