Drechslerella dactyloides and Dactylaria brochopaga mediated structural defense in tomato plants pre-challenged with Meloidogyne incognita

Journal Pre-proofs Drechslerella dactyloides and Dactylaria brochopaga mediated structural defense in tomato plants pre-challenged with Meloidogyne incognita Udai B. Singh, Shailendra Singh, Deepti Malviya, Rajan Chaurasia, Pramod K. Sahu, Sushil K. Sharma, A.K. Saxena PII: DOI: Reference:

S1049-9644(19)30271-3 https://doi.org/10.1016/j.biocontrol.2020.104202 YBCON 104202

To appear in:

Biological Control

Received Date: Revised Date: Accepted Date:

4 April 2019 9 January 2020 13 January 2020

Please cite this article as: Singh, U.B., Singh, S., Malviya, D., Chaurasia, R., Sahu, P.K., Sharma, S.K., Saxena, A.K., Drechslerella dactyloides and Dactylaria brochopaga mediated structural defense in tomato plants prechallenged with Meloidogyne incognita, Biological Control (2020), doi: https://doi.org/10.1016/j.biocontrol. 2020.104202

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Elsevier Inc. All rights reserved.

Drechslerella dactyloides and Dactylaria brochopaga mediated structural defense in tomato plants pre-challenged with Meloidogyne incognita Udai B. Singh1*, Shailendra Singh1†, Deepti Malviya1†, Rajan Chaurasia2†, Pramod K. Sahu, Sushil K. Sharma1, and A.K. Saxena1

1Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-National Bureau of Agriculturally Important Microorganisms, Kushmaur, Maunath

Bhanjan 275 103, India 2Department

of Botany, Faculty of Sciences, Banaras Hindu University, Varanasi 221 005, India

† These authors have contributed equally. *Corresponding author: Email Address: [email protected] (Udai B. Singh) Tel.: +91-547-2530080, Fax: +91-547-2530358

Abstract The present study was undertaken with the objectives to study Drechslerella dactyloides NDAd-05 and Dactylaria brochopaga NDDb-15 mediated induction of structural defense responses leading to lower disease development and promoting growth in tomato pre-challenged with Meloidogyne incognita. The potential strains D. dactyloides NDAd-05 and D. brochopaga NDDb-15 were taken from Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-National Bureau of Agriculturally Important Microorganisms, Kushmaur, Maunath Bhanjan, India. In vitro root colonization assay was performed using D. dactyloides NDAd-05 and D. brochopaga NDDb-15 in sand:soil culture (2:1) and significant tomato root colonization by NDAd-05 and NDDb-15 was recorded. The study elucidated multifarious effects of D. dactyloides NDAd-05 and D. brochopaga NDDb-15 when inoculated either individually or in combination in tomato plants pre-challenged with M. incognita. Additionally, D. dactyloides NDAd-05 and D. brochopaga NDDb-15 increased antioxidant as well as biocontrol activities significantly in tomato against M. incognita. Microscopic visualization of H2O2 and superoxide radicals in tomato leaves further corroborated the above findings. Further, inoculation of D. dactyloides NDAd-05 and D. brochopaga NDDb-15 activated the phenylpropanoid pathway in roots leading to increase cell wall lignifications and pectin deposition in tomato roots in addition to direct trapping and parasitizing of juveniles and adults of M. incognita. From the results it can be concluded that increased cell wall lignifications and pectin deposition probably restricted the entry of nematodes and ultimately decreased the M. incognita population in tomato roots. It was also observed that plants treated with bioagents individually or in combination modulated the phenotypical alterations and assisted plant growth promotion. This might be due to the interaction-dependent modulation of physio-biochemical pathways in the tomato plants which ultimately reduced the inoculum potential and disease intensity of M. incognita.

Keywords: Meloidogyne incognita, Drechslerella dactyloides; Dactylaria brochopaga; Induced systemic resistance; Root-knot disease 1. Introduction Root-knot nematodes (RKNs) are sedentary endoparasites belong to the genus Meloidogyne. RKNs are obligate plant parasite in nature. Being ubiquitous in nature, RKNs parasitize the roots of thousands of plant species, including monocotyledonous, dicotyledonous, herbaceous and woody plants (Singh et al., 2013a,b; Singh et al., 2019). Nearly 2,000 plants species are susceptible to root-knot nematodes worldwide. RKNs cause approximately 5% of the global crop loss (Sasser and Carter, 1985). The genus Meloidogyne includes more than 90 species with several physiological races (Walia and Bajaj, 2003). Initially, second stage juveniles (J2s) invade the elongation region of the root and migrate inside the root system until they become sedentary. The parenchyma cells of the plant roots near the head of the J2s become multinucleate and develop into feeding cells known as giant cells, from which the J2 and later the adults feed (Sijmons et al., 1994; Hussey and Grundler, 1998). Simultaneously, with the giant cell formation, excessive cell division takes place in the surrounding root cells and knot development is initiated, in which the developing juveniles are embedded and thereby deprive nutrients and photosynthates to the infested plants (Sijmons et al., 1994; Hussey and Grundler, 1998; Su et al., 2017). Owing to their broad host range, and the quantity of damage they cause, RKNs have become major menace and difficult to manage pest of vegetable crops worldwide. In vegetable crops, the yield losses by RKN species are estimated between 28 and 47%, especially due to the M. incognita infestation (Anonymous, 1986; Upadhyay and Dwivedi, 2008). Several pesticides including soil fumigants have been reported to reduce infestation of M. incognita (Singh et al., 2019). However, due to their detrimental effects on non-target micro-flora and fauna they have been least preferred in the current scenario of organic agriculture (Gupta et

al., 2017a, b). Therefore, as alternative of toxic chemical pesticides, several bioagents such as Purpureocillium lilacinus, Arthrobotrys oligospora, Pochonia chlamydosporia, Dactylaria spp., Monacrosporium spp., Drechslerella dactyloides, Syncephalastrum racemosum, Hirsutella spp., Duddingtonia spp. and Trichoderma spp. have been tested and used to manage RKNs worldwide (Singh et al., 2013a, b; Wang et al., 2014; Huang et al., 2014; Gupta et al., 2015a, b; Singh et al., 2017). Among the bioagents, nematophagous fungi have widely been used across the world for their high effectiveness (Ciancio et al., 2016; Su et al., 2017; Gupta et al., 2017a,b; Singh et al., 2019). They develop specific capturing structures (adhesive networks, constricting rings, adhesive knobs, etc.) to trap nematodes at different stages (Jansson and Lopez-Llorca, 2001; NordbringHertz et al., 2006; Singh et al., 2012a, b, c; Singh et al., 2013a,b). In our previous study, we demonstrated induction and differential bioaccumulation of defence related biomolecules under the influence of nematode trapping fungi A. oligospora (Singh et al., 2013a), D. brochopaga and D. dactyloides (Singh et al., 2019). Moreover, predacity of M. incognita by D. dactyloides or D. brochopaga depends on virulence of the strain, nematode species, nematode population and number of traps induced in presence of nematodes (Singh et al., 2019). Singh et al. (2019) demonstrated that these two fungi effectively parasitized nematodes present in the rhizosphere of tomato (Solanum lycopersicum L.) and protected the plants both through direct and/or indirect mechanisms. However, there is lack of information on interaction-dependent activation of structural defence responses, deactivation of super oxides and reactive oxygen species leading to systemic resistance in plants pre-challenged with M. incognita. Further, how the bioagents D. dactyloides and D. brochopaga influence the host defence responses is also not clearly understood. Considering the importance of the problem and potentiality shown by D. dactyloides NDAd-05 and D. brochopaga NDDb-15 in our previous studies, the present study was undertaken with the objectives to study D. dactyloides NDAd-05 and D. brochopaga NDDb-15 mediated induction

of structural defense responses in tomato pre-challenged with M. incognita leading to induced systemic resistance and plant growth promotion in tomato. 2. Materials and Methods 2.1. Fungal bioagents and inocula of Meloidogyne incognita The fungal strains D. dactyloides NDAd-05 and D. brochopaga NDDb-15 were obtained from Plant-Microbe Interaction and Rhizosphere Biology Lab, ICAR-National Bureau of Agriculturally Important Microorganisms, Kushmaur, Maunath Bhanjan, India. Fungal cultures were maintained on corn meal agar (CMA) medium (HiMedia, India) by sub-culturing at 15 days interval. The vermi-based bioformulations of D. dactyloides NDAd-05 and D. brochopaga NDDb-15 was prepared by following the steps given by Singh et al. (2019). The colony forming unit of the developed bioformulation of D. dactyloides NDAd-05 and D. brochopaga NDDb-15 was 1.45×106 cfu g-1 and 2.02×106 cfu g-1, respectively. Freshly M. incognita infected tomato plants were uprooted from sick pots maintained at ICAR-NBAIM, Kushmaur and farmers’ fields. The second stage juveniles (J2s) were extracted as per the method described by Singh et al. (2019) and the numbers of J2s were counted with the help of nematode counting dish. These freshly extracted J2s were used as inoculums in the subsequent pot experiments. 2.2. Planting materials Tomato seedlings (cv. Dev) were raised in sterile potting mixture of sand, soil and vermicompost (1:1:1) in a nethouse at ICAR-NBAIM, Kushmaur, Maunath Bhanjan following the standard agronomic practices. Forty day-old tomato seedlings were uprooted gently and used for pot experiments. Two seedlings were transplanted to each pot containing 5 kg of experimental soil with or without inoculation of D. dactyloides

NDAd-05 and D. brochopaga NDDb-15 under nethouse conditions. The pot experiments were conducted during October to December with relative humidity of 80-90% and average mean temperature of 23 C (17-31C) under 11/-13 h light/dark photoperiod. 2.3. Root colonization assay The strains D. dactyloides NDAd-05 and D. brochopaga NDDb-15 were tested for their rhizosphere colonization ability following the method described by Persson and Jansson (1999) with slight modifications. Five tomato seedlings were transplanted in a pot filled with pre-sterilized peat containing 20% sand. The bioagents D. dactyloides NDAd-05 and D. brochopaga NDDb-15 were added in the four corners about 2 cm from the seedlings at a depth of 2.5 cm. D. dactyloides NDAd-05 and D. brochopaga NDDb-15 were introduced into the soil as grain-based formulation (0.5 g at each place). Root colonization was observed under compound light microscope (Olympus BX 41). Furthermore, fungal growth in the rhizosphere and non-rhizosphere was studied as per the method of Persson and Jansson (1999) at 15, 30, 45 and 60 days of transplanting. 2.4. Experimental design and nethouse experiments Nethouse experiments were conducted to evaluate the bio-efficacy of vermi-based formulations of D. dactyloides NDAd-05 and D. brochopaga NDDb-15 against root knot diseases in tomato at ICAR-NBAIM, Maunath Bhanjan, India. There were six treatments and each treatment consisted of ten replications in the nethouse experiments. The treatments were: T-1: M. incognita, T-2: M. incognita and D. dactyloides NDAd-05, T-3: M. incognita and D. brochopaga NDDb-15, T-4: M. incognita, D. dactyloides NDAd-05 and D. brochopaga NDDb-15, T-5: M. incognita with carbofuran, and T-6: control (untreated). The experiments were repeated twice. Further, the experimental soil contained in the pots was mixed with the J2s of the M. incognita at 2500 J2s kg-1 of soil. On the second day, 20g vermi-based formulations of D. dactyloides NDAd-05 and D. brochopaga

NDDb-15 alone and in combination (1:1 ratio) was inoculated and properly mixed in the pots containing nematode infested soil. The cfu count of the selected nematode trapping fungi when inoculated in the combination was 1.72×106 cfu g-1. The untreated soil with equivalent amount of nonfortified vermi-compost served as control (T6). However, soil with carbofuran (at 2 kg a.i. ha-1) and non-fortified vermicompost served as chemical control (T5). After inoculation of bioagent(s), tomato seedlings (two) were transplanted into each pot in the evening and watered daily using sterile distilled water to maintain the moisture at field capacity. 2.5. Sampling and analysis 2.5.1. Detection and visualization of superoxide radicals (O2−) and H2O2 Plant leaves were sampled randomly from each treatment at 45 days of transplanting and used for microscopic localization of superoxide radicals (O2−) and H2O2. For detection of O2−, nitroblue tetrazolium (NBT; HiMedia, India) was used (Rao and Davis, 1999) and its occurrence was detected as a blue color spot in the leaves. However, visualization of H2O2 was carried as per the method described by Sakamoto et al. (2008). In brief, the intracellular H2O2 was detected by using 3, 3-diaminobenzidine (DAB; HiMedia, India). DAB was dissolved in H2O and adjusted to pH 3.8 with KOH. Leaf strips were bleached in acetic acid-glycerol-ethanol solution (1/1/3, v/v/v) at 100 °C for 5 min, transferred in the staining solution and infiltrated under vacuum with 1.25 mg mL-1 DAB staining solution and then observed under a stereomicroscope (Model: SZX 7, Olympus, Japan). Images were taken with a digital camera (Nikon, Japan). Under stereoscope, presence of H2O2 was visualized as a brown color deposition in the plant leaves due to DAB polymerization. 2.5.2. Effects of bioinoculants on activation and accumulation of H2O2 and enzymes

Effect of D. dactyloides and D. brochopaga inoculation on H2O2 content, phenylalanine ammonia lyase (PAL), peroxidase, chitinase and superoxide dismutase (SOD) activity in the tomato leaves pre-challenged with M. incognita in pot experiments at 30 days of transplanting under nethouse condition was observed. For quantitative estimation of H2O2, leaf samples (150 mg) were collected at 30 days of transplanting and homogenized in 0.05 mM phosphate buffer (pH 7.2). The supernatant was collected after centrifugation at 10000 g for10 min at 4 °C. The H2O2 content in the plant leaves was measured at 405nm wavelength using UV Vis Spectrophotometer (Model: UV-2450, Shimadzu) as per the method described by Yang et al. (2018). Further, activities of PAL and peroxidase were measured in the plant leaves following the protocols described by Sadasivam and Manickem (1996). However, chitinase and SOD activities in tomato leaves were measured spectrophotometrically as per the method described by Thimmaiah (2012). 2.5.3. Histo-pathological study Tomato plants were uprooted from each treatment at 45 days of transplanting for histo-pathological study. Transverse sections of roots were cut manually and selected fine sections were stained with the counter stain safranin. Differential staining of lignin was carried out as per the method described by Jensen (1962). Briefly, the fine sections were fixed in ethanol (gradients 10, 25, 50, 75, 80, and 95% v/v) and mounted on a glass slide in saturated aqueous solution of phloroglucinol in 20% HCl and the sections from each treatment were observed under a light microscope (Model: BX 41, Olympus, Japan).Positive lignin staining was indicated by red-violet colour under compound light microscope. The intensity of red-violet colour reflected the amount of lignin present in root cells.

Ruthenium red was used to localize and visualize pectin deposition in tomato roots treated with the bioagents alone or in combination. Briefly, selected sections were placed in 0.02% aqueous ruthenium red solution for 30 min, washed gently to remove the excess stain, mounted in glycerol and visualized under light microscope. Development of pink colour after staining indicated presence of unesterified (acidic) pectin. The intensity of red colour reflected the amount of pectin present in root cells. Further, quantitative estimation of lignin and uronic acid contents was done in tomato roots after 45 days of transplanting. The root samples were collected from each treatment, washed in running tap water and air dried. Lignin content in the root samples was estimated as per the protocol defined by Yang et al. (2018). However, pectin uronic acid content was measured spectrophotometrically according to Blumenkrantz and AsboeHansen (1973) with slight modifications (Zhu et al., 2015). Galacturonic acid (Sigma-Aldrich) was used as standard and absorbance was measured at 520 nm. 2.5.4. Plant growth promotion and disease dynamics Five plants from each treatment were randomly sampled to find out the average numbers of root galls developed per seedling, number of J2s present per root system, shoot and root length and fresh weight of shoots and roots per plant at 45 and 60 days after transplanting. Further, trapped formed in the rhizosphere soil and captured nematodes were recorded at 45 days of transplanting. Final population of J2s in the root galls was counted according to Bridge et al. (1981). Briefly, infected roots along with galls were stained in boiling 0.1% (w/v) acid fuchsin in glycerol, lactic acid and distilled water (1:1:1). Stained roots were macerated in distilled water and number of J2s, and adult male and females were counted under light stereomicroscope.

2.6. Statistical analysis The laboratory experiments were laid out in Completely Randomized Design in five replications, whereas, the nethouse experiments were laid out in Randomized Block Design in ten replications. All experiments were repeated twice to see consistency of the results. Data obtained were subjected to analysis of variance (ANOVA) using statistical package for Social Sciences Version 16.0 (SPSS 16.0) programme. Means were compared by least significant difference test (LSD test) at p <0.05.

3. Results 3.1. Root colonization assay Percent root colonization was recorded at 45 days of transplanting of tomato seeds. Results showed that 59.50% of roots were colonized in case of D. dactyloides NDAd-05, whereas, 55.25% of roots were colonized by D. brochopaga NDDb-15 under controlled conditions (Fig. 1 a-d). Both the strains colonized the tomato roots, rhizosphere and non-rhizosphere soil. Maximum propagules were recorded in the rhizosphere soil treated with D. dactyloides NDAd-05 (112.45 g-1 soil) compared to non-rhizosphere soil (85.25 g-1 soil) at 60 days of planting (Fig. 2a). However, in case of D. brochopaga NDDb-15, the rhizosphere soil contained 78.96 propagules g-1 soil (Fig. 2b). 3.2. Effect of bioinoculants on O2− and H2O2 generation in tomato leaves Stereoscopic visualization of superoxide radicals in plant leaves revealed that under pathogen challenged conditions superoxide content was maximally localized in the petioles near veins and midrib (Fig. 3). Plants treated with the bioagents individually or in combination exhibited considerably decreased superoxide levels in the mature leaves followed by those under carbofuran treatment. Attenuation of superoxide levels was observed as a blue formazan which is the outcome of NBT dye and superoxides interactions (Fig. 3). Similarly, H2O2 accumulation was observed as brown polymerization product of DAB staining. The bioinoculant(s) treatment significantly reduced brown discolouration compared to M. incognita treated plants. However, least amount of brown pigmentation was observed in the control plants (Fig. 4). 3.3. Effects of bioinoculants on activation and accumulation of H2O2 and enzymes

Accumulation and activity of defense related biomolecules and enzyme activity in the plant system is known to be a good predictor for stress signals. Results indicated that maximum H2O2 was recorded in the leaves of plants inoculated with M. incognita alone (26.32 mmol mg-1 protein) followed by plants treated with both D. dactyloides NDAd-05 (20.95 mmol mg-1 protein) and D. brochopaga NDDb-15 (19.47 mmol mg-1 protein). The least amount of H2O2 was recorded in the control plants (Table 1). Foliar PAL and peroxidase activities were significantly related to induced systemic resistance. The results revealed that 2-fold increment in the activities of PAL and peroxidase was recorded in the plants inoculated with D. dactyloides NDAd-05 and D. brochopaga NDDb-15 in combination compared to M. incognita alone treated plants. Additionally, no significant difference in PAL activity was recorded in the plants inoculated with D. dactyloides NDAd-05 and D. brochopaga NDDb-15 under pathogenic stress, but significant difference was recorded for peroxidase activity in the same treatments. The data indicated that the phenylpropanoid pathway might have been activated and played a key defensive role in tomato under stress of M. incognita (Table 1). A similar trend was recorded in case of chitinase and SOD activities as well in the tomato plants (Table 1). 3.4. Effect of bioagents colonization on lignin and pectin deposition Plants treated with D. dactyloides NDAd-05 and D. brochopaga NDDb-15 alone or in combination showed significant variations in lignin deposition as indicated by intensity of the red colour. Maximum and uniform lignin deposition in vascular bundles and pericycle were observed in the plant roots co-inoculated with D. dactyloides NDAd-05 and D. brochopaga NDDb-15 under pathogenic stress conditions followed by plants inoculated with either of the bioagents individually and carbofuran treated plants (Fig. 5). Lignin deposition was least in unchallenged control plants. Observations from this experiment emphasized the role of bioagents in strengthening the physical defense response in tomato against M.

incognita challenge (Fig. 5). A similar trend was also observed in case of pectin deposition in the root tissues (Fig. 6). The roots stained with ruthenium red exhibited pink colour indicating the presence of demethylesterified pectin. The pink colour was observed mostly in the middle lamella and the secondary fibre cell wall. Pectin was present in all the samples observed. A significant difference was observed in the distribution of demethylesterified pectin across the treatments. The higher stain intensity (degree) showed high pectin content in the root tissues. Results showed significantly weaker signal of pink colour in plants inoculated with the individual bioagents compared to plants co-inoculated with both the bioagents (Fig. 6). This was further confirmed with the quantitative estimation data. The quantitative estimation showed lignin and pectin uronic acid content in D. dactyloides NDAd-05 and D. brochopaga NDDb-15-induced tomato roots was significantly higher than that of the pathogen alone and untreated control plants at 45 days of transplanting. 1.5-fold increase in the lignin and pectin uronic acid content was recorded in tomato roots treated with both the bioagents compared to tomato plants treated with M. incognita alone and untreated control (Table 2). The lignin content of plant roots treated with D. dactyloides NDAd-05 and D. brochopaga NDDb15 individually did not differ significantly. However, significant difference was recorded for pectin uronic acid content (Table 2). The least amount of lignin and pectin uronic acid was measured in untreated control plants. 3.5. Effect of bioinoculants on plant growth and disease dynamics Root colonization by D. dactyloides NDAd-05 and D. brochopaga NDDb-15 (alone or in combination) significantly enhanced plant growth parameters and decreased root-knot disease compared to the pathogen inoculated plants in nethouse experiments (Fig. 7 and Table 3). Figure 7 clearly showed that D. dactyloides NDAd-05 and D. brochopaga NDDb-15 colonized in the rhizosphere soil, produced the trapping devices and

captured J2s present in the soil. Further, results revealed that plants co-inoculated with the test RKN, D. dactyloides NDAd-05 and D. brochopaga NDDb-15, the number of root galls, and J2sper plant were significantly reduced (50 %) compared to M. incognita alone inoculated plants at 45 and 60 days after transplanting (Table 3). However, the treatment involving carbofuran showed minimum number of root galls, and J2s per plant at 45 and 60 days of transplanting (Table 3). Correspondingly, maximum increase in shoot and root length with >3-folds was recorded in the plants co-inoculated with D. dactyloides NDAd-05 and D. brochopaga NDDb-15 compared to the M. incognita inoculated plants at 45 and 60 days of transplanting (Table 3). Even though, significant increments in the shoot and root length was recorded in the plants inoculated with either of the two bioinoculants compared to M. incognita inoculated (T1) plants. A similar trend was also recorded in case of fresh weight of shoot. However, maximum root weight was recorded in the plants treated with M. incognita alone due to gall development at 45 and 60 days of transplanting in the nethouse experiments (Table 3). 4. Discussion Meloidogyne incognita is a sedentary endoparasite and obligate in nature. Generally, J2s infect the plant roots and move inside the plant roots and later surrounding tissues are being converted into gall/knot. Although cosmopolitan in occurrence, devastating infection is mainly reported from countries with warm and humid climate. Looking into the great importance of the pathogen, the present study demonstrated the significance of D. dactyloides NDAd-05 and D. brochopaga NDDb-15 mediated induction of structural defense response in the plants pre-challenged with M. incognita in lowering disease development and increasing plant growth in tomato. In the current study, it was observed that D. dactyloides NDAd05 and D. brochopaga NDDb-15 colonized the tomato roots, rhizosphere and non-rhizosphere soil. Further, this study also showed that both

bioagents formed trapping structures i.e. three-celled constricting rings in the presence of the nematodes and sufficient capturing was recorded in the rhizosphere soil. Nematode-induced formation of different types of trapping structures by the nematophagous fungi is well documented (Singh et al., 2012a, b, c; Singh et al., 2013a,b; Kumar et al., 2015, Singh et al., 2019). These trapping devices are being used to capture nematodes which served as a source of nutrition for invading fungi. Thus, the fungi have a two-way relationship with the nematodes: first, the nematodes induced formation of trapping structures in fungi to capture nematodes; and, second, the nematodes serve as an additional source of nutrition to the invading fungi for their growth and development (Kumar et al., 2015; Sellitto et al., 2016; Singh et al., 2019). Results of the present study clearly indicate that D. dactyloides NDAd-05 and D. brochopaga NDDb-15 capture juveniles of the M. incognita directly and decreased its population in the root system and rhizosphere soil leading to substantial decrease in gall development in tomato roots. In our previous studies, we have demonstrated D. dactyloides NDAd-05 and D. brochopaga NDDb-15 mediated site-specific accumulation of defense related mediator molecules and activation of enzymes such as chitinase, β 1, 3-glucanase, phenylalanine ammonia lyase (PAL) and peroxidase in the M. incognita-tomato pathosystem (Singh et al., 2019). However, involvement of these two bioagents D. dactyloides NDAd-05 and D. brochopaga NDDb-15 in activation of structural defense and antioxidant activities with reduced toxic effects of superoxides generated during pathogen infection in tomato plants pre-challenged with M. incognita has not been studied in detail. This gives insights to investigate the D. dactyloides and D. brochopaga mediated physiobiochemical mechanisms in root-knot-tomato pathosystem in a deeper way. Therefore, in the present study, we have demonstrated that root colonization by D. dactyloides NDAd-05 and D. brochopaga NDDb-15 activated the antioxidant activities in tomato leaves and site-specific activation of lignin and pectin deposition in the root tissues leading to structural defense against infection caused by M. incognita. In contrast,

histo-pathological study showing higher lignification in the plant root treated with both the bioagents at 45 days after inoculation may be due to passive transport of the monolignols and/or synthesis of lignin in the root apoplast. This was also confirmed by the quantitative estimation of lignin in the root tissues (Table 2). Lignin is the product of phenylpropanoid pathways constitutively present in higher plants. Its content, composition and nature may change when plants are exposed to various abiotic and biotic stresses (Hahlbrock and Scheel, 1989; Ali and McNear, 2014; Gomes et al., 2017; Leonetti et al., 2017). Lignin plays an important role in resisting pathogen infection in plants as it provides physical barriers that make the cell walls more resistant to mechanical pressure during pathogen infection (Smit and Dubery, 1997; Bu et al., 2014). Our results clearly indicated that the cell wall in the bioagent-treated roots was obviously thickened compared to the pathogen alone treated plants (Fig. 5). High levels of lignin and cell wall thickness (as depicted in Fig. 5) could enhance the toughness and mechanical strength of the cell wall and reduce cell gaps (inter-cellular spaces), leading to resistance to pathogen infection (Smit and Dubery, 1997; Xu et al., 2011). In addition, significantly higher accumulation of pectin in the plant roots co-inoculated with D. dactyloides NDAd-05 and D. brochopaga NDDb-15, further strengthens the possibilities of reduced infection and movement of the J2s in the root tissues. Pectin is responsible for cell wall adhesion between cells. It has been reported that increased lignification and pectin deposition are often recorded in response to attack by invader and is believed to represent one of the mechanisms adopted by plants as physio-biochemical barrier to restrict entry of the invader due to non-degradable and antimicrobial nature of lignin (Dixon and Paiva, 1995; Rogers and Campbel, 2004; Patel et al., 2017). In our previous study, we showed manifold increase in PAL and peroxidase activities in plant roots and shoots co-inoculated with both the bioagents which are the key enzymes of the lignin synthesis pathway (Singh et al., 2019). Results also showed variation in the lignin deposition in the tomato roots treated with either of the two bioagents under M.

incognita stress condition. This variation is attributed to the role of the bioagents in different combinations in triggering the phenylpropanoid pathway that eventually leads to the lignification process to an enhanced level. Moreover, we observed better and extended lignification in the xylem and phloem cells of the plants treated with both the bioagents. To our knowledge, this is the first report depicting the interactive role of D. dactyloides and D. brochopaga in conferring physio-biochemical strength to the tomato roots in the tomato-M. incognita pathosystem. Several researchers reported that genes coding for PAL, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase showed elevated expression under pathogenic stress condition and PAL activity was also enhanced (Sarma et al., 2015; Kumar et al., 2017; Święcicka et al., 2017; Manikandan et al., 2018). However, information on the role of NTF in enhancing the physical strength of plant’s cell wall towards a pathogenic stress is adequate and thus, findings of the current investigation help in understanding the role of such microbes in biological control of soilborne pathogens especially in tomato-M. incognita pathosystem. This is first report of NTF mediated induction of structural defense in tomato. 5. Conclusion RKNs especially M. incognita cause severe damage to the tomato crop resulting in huge loss. Nematode trapping fungi (NTF) are important biocontrol agents for RKNs. As observed in the present study, the growth and development of M. incognita in tomato roots was least in the coinoculation treatment. Less disease development in the present study may be attributed to activation of the phenylpropanoid pathway in the plants treated with D. dactyloides and D. brochopaga in addition to direct trapping for control of M. incognita. The effect was further improved by coinoculation of two NTF namely D. dactyloides and D. brochopaga which demonstrates their compatibility with each other not only in strengthening the plant against the pathogenic invasion but also supporting the plant growth. These two bioagent(s) applied individually or in combination

reprogram biosynthesis and accumulation of lignin and pectin in the root tissues leading to reduction in disease development under pathogenic stress conditions. Conflicts of Interest The authors have declared no conflict of interest. Acknowledgement We would like to express our special thanks to Dr. R.K. Singh and Dr. B.K. Sarma, Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, BHU, Varanasi for providing technical support to carry out a part of this research work. The authors wish to thank to P.K. Sharma, Harsh V. Singh and Manish Roy, ICAR-NBAIM, Kushmaur, Maunath Bhanjan, India for technical assistance in the microscopy and preparation of manuscript. Our special thanks goes to Science and Engineering Research Board (SERB), Department of Science and Technology, Ministry of Science & Technology, Government of India for providing financial support to Udai B. Singh under Fast Track Scheme for Young Scientist (SR/FT/LS-42/2012) to carry out the research work. References Ali, M.B., McNear D.H., 2014. Induced transcriptional profiling of phenylpropanoid pathway genes increased flavonoid and lignin content in Arabidopsis leaves in response to microbial products. BMC Plant Biol. 14, 84. Anonymous, 1986. Annual Report. Plant Pathology Division, BARI, Joydebpur, Gazipur. p. 68. Blumenkrantz, N., Asboe-Hansen, G., 1973. New method for quantitative determination of uronic acids. Analytical Biochemistry 54, 484–489.

Bridge, J., Page, S.J., Jordan, W., 1981. An improved method for staining nematodes in roots. UK, Report Rothamsted Experimental Station. p. 171. Bu, B., Qiu, D., Zeng, H., Guo, L., Yuan, J., Yang, X., 2014. A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton. Plant Cell Rep. 33(3), 461–70. Ciancio, A., Colagiero, M., Pentimone, I., Rosso, L.C., 2016. Formulation of P. chlamydosporia for plant and nematode management. In: Arora N., Mehnaz S., Balestrini R. (eds) Bioformulations: for Sustainable Agriculture. Springer, New Delhi. pp. 177-197. Dixon, R.A., Paiva, N.L., 1995. Stress-lnduced phenylpropanoid metabolism. The Plant Cell 7, 1085-1097. Gomes, E.V., Ulhoa, C.J., Cardoza, R.E., Silva, R.N., Gutiérrez, S., 2017. Involvement of T. harzianum Epl-1 protein in the regulation of Botrytis virulence and tomato defense-related genes. Front. Plant Sci. 8, 880. Gupta, R. et al., 2015a. Exploitation of microbes for enhancing bacoside content and reduction of M. incognita infestation in B. monnieri L. Protoplasma 252, 53–61. Gupta, R. et al., 2017a. Microbial modulation of bacoside a biosynthetic pathway and systemic defense mechanism in B. monnieri under M. incognita stress. Sci. Rep. 7, 41867. Gupta, R., Saikia, S.K., Pandey R., 2015b. Bioconsortia augments antioxidant and yield in Matricaria recutita L. against M. incognita (Kofoid and White) Chitwood infestation. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.87(2), 335-342.

Gupta, R., et al., 2017b. Microbial interference mitigates M. incognita mediated oxidative stress and augments bacoside content in B. monnieri L. Microbiol. Res. 199, 67–78. Hahlbrock, K., Scheel, D., 1989. Physiology and molecular Biology of phenylpropanoid Metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 347-69. Huang, W.K., Sun, J.H., Cui, J.K., Wang, G.F., et al., 2014. Efficacy evaluation of fungus Syncephalastrum racemosum and nematicide avermectin against the RKN M incognita on cucumber. PLoS One 9e89717. Hussey, R.S., Grundler, F.M., 1998. Nematode parasitism of plants. Physiology and Biochemistry of Free-Living and Plant Parasitic Nematodes. CAB International Press, Oxford, pp. 213-243. Jansson, H.B., Lopez-Llorca, L.V., 2001. Biology of nematophagous fungi. In: Misra, J.K., Horn, B.W. (Eds.) Mycology: Trichomycetes, other fungal groups and mushrooms. Enfield, M.A., Science Publishers, USA. p. 145–73. Jensen, W.A., 1962. Botanical Histo-chemistry: Principles and Practices. London: WH Freeman and Co. San Francisco and London. p. 408. Kumar, D., Maurya, N., Kumar, P., Singh, H., Addy, S.K., 2015. Assessment of germination and carnivorous activitiesof a nematode-trapping fungus Arthrobotrys dactyloides in fungistatic and fungicidal soil environment. Biol. Control 82, 76–85. Kumar, R., Bohra, A., Pandey, A.K., Pandey, M.K., Kumar, A., 2017. Metabolomics for plant improvement: Status and Prospects. Front. Plant Sci. 8, 1302.

Leonetti, P., Zonno, M.C., Molinari, S., Altomare, C., 2017. Induction of SA-signaling pathway and ethylene biosynthesis in T. harzianum-treated tomato plants after infection of the root-knot nematode M. incognita. Plant Cell Reports 36, 621-631. Manikandan, R., et al., 2018. Comparative proteomic analysis of different isolates of F. oxysporum f.sp. lycopersici to exploit the differentially expressed proteins responsible for virulence on tomato plants. Front. Microbiol. 9, 420. Nordbring-Hertz, B., Jansson, H.B., and Tunlid, A., 2006. Nematophagous Fungi. Encyclopedia of life sciences. p. 1-11, Patel, J.S., Kharwar, R.N., Singh, H.B., Upadhyay, R.S. and Sarma, B.K., 2017. T. asperellum (T42) and P. fluorescens (OKC)-Enhances resistance of pea against E. pisi through enhanced ROS generation and lignifications. Front. Microbiol. 8, 306. Persson, C., Jansson, H.B., 1999. Rhizosphere colonization and control of Meloidogyne spp. by nematode-trapping fungi. J. Nematol. 31, 164– 171. Rao, M.V., Davis, K.R., 1999. Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid. Plant J. 17, 603–614. Rogers, L.A., Campbel, M.M., 2004. The genetic control of lignin deposition during plant growth and development. New Phytol. 164, 17-30. Sadasivam, S., Manickam, A., 1996. Biochemical Methods.New Age International (P) Ltd. New Delhi, India. p. 256. Sakamoto, M., Munemura, I., Tomita, R., Kobayashi, K., 2008. Involvement of hydrogen peroxide in leaf abscission signaling, revealed by analysis with an in vitro abscission system in Capsicum plants. Plant J. 56, 13–27.

Sarma, B.K., Yadav, S.K., Singh, S., Singh, H.B., 2015. Microbial consortium-mediated plant defense against phytopathogens: Readdressing for enhancing efficacy. Soil Biol. Biochem.87, 25-33. Sasser, J.N., Carter, C.C., 1985. Overview of the international Meloidogyne project 1975-1984. Sellitto, V.M., Curto, G., Dallavalle, E., Ciancio, A., Colagiero, M., Pietrantonio, L., Bireescu, G., Stoleru, V., Storari, M., 2016. Effect of Pochonia chlamydosporia-based formulates on the regulation of root-knot nematodes and plant growth response. Front. Life Sci. 9, 177-181, Sijmons, P.C., Atkinson, H.J., Wyss, U., 1994. Parasitic strategies of root nematodes and associated host cell responses. Ann. Rev. Phytopath 32(1), 235-259. Singh, U.B., Sahu, A., Sahu, N., Singh, B.P., et al., 2013a. Can endophytic Arthrobotrys oligospora modulate accumulation of defence related biomolecules and induced systemic resistance in tomato (Lycopersicon esculentum Mill.) against root knot disease caused by Meloidogyne incognita. Appl. Soil Ecol. 63, 45-56. Singh, U.B., Sahu, A., Sahu, N., Singh, R.K., et al., 2012a. Co-inoculation of D. brochopaga and M. eudermatum affects disease dynamics and biochemical responses in tomato (L. esculentum Mill.) to enhance bioprotection against M. incognita. Crop Protect.35, 102-09. Singh, U.B., Sahu, A., Singh, R.K., Singh, D.P., et al., 2012b. Evaluation of biocontrol potential of A. oligospora against M. graminicola and R. solani in Rice (Oryza sativa L.). Biol. Control 60, 262-70.

Singh, Udai B., Sahu, A., Sahu, N., Singh, R.K., Renu, Singh, D., Singh, B.P., Jaiswal, RK, et al.,2013b. Nematophagous fungi: Catenaria anguillulae and Dactylaria brochopaga from seed galls as potential biocontrol agents of Anguina tritici and Meloidogyne graminicola in wheat (Triticum aestivum L.). Biological Control 67, 475–482 Singh, Udai B., Singh, S., Khan, W., Malviya, D., Sahu, Pramod K., et al., 2019. Drechslerella dactyloides and Dactylaria brochopaga mediated induction of defense related mediator molecules in tomato plants pre-challenged with Meloidogyne incognita. India Phytopath. Doi: 10.1007/s42360-019-00132-x Singh, Udai.B., Sahu, A., Sahu, N., Singh, R.K., et al., 2012c. A. oligospora mediated biological control of diseases of tomato (L. esculentum Mill.) caused by M. incognita and Rhizoctonia solani. J. Appl. Microbiol.114, 196–208. Smit, F., Dubery, I.A., 1997. Cell wall reinforcement in cotton hypocotyls in response to a Verticillium dahliae elicitor. Phytochemistry 44(5), 811–815. Su, L., Shen, Z., Ruan, Y., Tao, C., Chao, Y., Li, R., Shen, Q., 2017. Isolation of antagonistic endophytes from banana roots against Meloidogyne javanica and their effects on soil nematode community. Front. Microbiol. 8, 2070. Święcicka, M., Skowron, W., Cieszyński, P., Dąbrowska-Bronk, J., Matuszkiewicz, M., Filipecki, M., Koter, M.D., 2017. The suppression of tomato defence response genes upon potato cyst nematode infection indicates a key regulatory role of miRNAs. PlantPhysiol. Biochem. 113, 51-55. Thimmaiah, S.R., 2012. Standard Methods of Biochemical Analysis. New Delhi: Kalyanipublishers. pp. 421–426.

Upadhyay, K.D., Dwivedi, K., 2008. A Text Book of Plant Nematology.Aman Publishing House, Meerut, India. p. 8. Walia, R.K., Bajaj, H.K., 2003. Textbook on Introductory Plant Nematology. Directorate of Information and Publication of Agriculture, Indian Council of Agricultural Research, KAB-II, New Delhi, India. p. 96. Wang, X., Li, Guo-Hong, Zou, Cheng-Gang, Ji, Xing-Lai, et al., 2014. Bacteria can mobilize nematode-trapping fungi to kill nematodes. Nat. Commun. 5, 5776. Xu, L., Zhu, L., Tu, L., Liu, L., Yuan, D., Jin, L., Long, L., Zhang, X. 2011. Lignin metabolism has a central role in the resistance of cotton to the wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry. J Exp Bot. 62(15), 5607– 5621. Yang, C., Liang, Y., Qiu, D., Zeng, H., Yuan, J., Yang, X., 2018. Lignin metabolism involves Botrytis cinerea BcGs1- induced defense response in tomato. BMC Plant Biol. 18, 103. doi:10.1186/s12870-018-1319-0 Zhu, X.F., Wang, Z.W., Wan, J.X., Sun, Y., Wu, Y.R., Li, G.X., Shen, R.F., Zheng, S.J., 2015. Pectin enhances rice (Oryza sativa) root phosphorus remobilization. J. Experimental Bot. 66, No. 3 pp. 1017–1024, 2015

Figure Legends Figure 1. The representative microphotographs showing colonization of tomato roots by selected strains of nematode trapping fungi (40X Magnification). (a-b) D. dactyloides NDAd-05 and (c-d) D. brochopaga NDDb-15. Arrows indicate fungal hyphae on the root surface. Figure 2. Colonization of tomato rhizosphere and non-rhizosphere soil by (a) D. dactyloides NDAd-05 and (b) D. brochopaga NDDb-15. Data are mean (n=5) and vertical bar lines represent standard error of mean. Figure 3. Microscopic detection of superoxide radical by NBT staining (5X) in leaves of tomato plants treated with nematode trapping fungi and pre-inoculated with M. incognita at 45 days of transplanting. NBT for localization of superoxides was indicated by arrows. Treatments were: 1- M. incognita, 2- M. incognita and D. dactyloides NDAd-05, 3- M. incognita and D. brochopaga NDDb-15, 4- M. incognita, D. dactyloides NDAd-05 and D. brochopaga NDDb-15, 5- M. incognita with carbofuran (chemical control), and 6- control (untreated). Figure 4. Histochemical detection and localization of H2O2 in tomato leaves by DAB staining (5X) treated with nematode trapping fungi and pre-inoculated with M. incognita at 45 days of transplanting. Localization of H2O2 was indicated by arrows. Treatments were: 1- M. incognita, 2- M. incognita and D. dactyloides NDAd-05, 3- M. incognita and D. brochopaga NDDb-15, 4- M. incognita, D. dactyloides NDAd-05 and D. brochopaga NDDb-15, 5- M. incognita with carbofuran (chemical control), and 6- control (untreated). Figure 5. Effect of D. dactyloides and D. brochopaga colonization on lignification in tomato root by histochemical staining (counter staining with safranin and Phloroglucinol-HCl) at 45 days of transplanting. Treatments were: 1- M. incognita, 2- M. incognita and D. dactyloides NDAd-05, 3- M. incognita and D. brochopagaNDDb-15, 4- M. incognita, D. dactyloides NDAd-05 and D. brochopagaNDDb-15, 5- M. incognita with carbofuran, and 6- control (untreated). Figure 6. Effect of D. dactyloides and D. brochopaga colonization on pectin deposition in tomato root pre-challenged with M. incognita by histochemical staining with ruthenium red at 45 days of transplanting, Treatments were: 1- M. incognita, 2- M. incognita and D. dactyloides NDAd-05, 3- M. incognita and D. brochopagaNDDb-15, 4- M. incognita, D. dactyloides NDAd-05 and D. brochopagaNDDb-15, 5- M. incognita with carbofuran, and 6- control (untreated). Figure 7. Formation of trapping structures and predation of root-knot nematodes juveniles in the rhizosphere soil of tomato pre-treated with D. dactyloides and D. brochopaga at 45 days of transplanting. Figure 6 (a-d) representing the formation of 3-celled constricting rings and captured nematode juveniles by D. dactyloides,

whereas, figure 6 (e-h) representing the formation of 3-celled constricting rings and captured nematode juveniles by D. brochopaga. Yellow and red arrows indicate formation of 3-celled constricting rings and captured nematode juveniles, respectively. Figure 1. (a)

(b)

(c)

(d)

Figure 2.

(a)

Propagulesof D. dactyloides (g-1 soil)

120 Non-rhizospheric soil 100

Rhizospheric soil

80 60 40 20 0 15

30

45 Days

(b)

60

Propagules of D. brochopaga (g-1 soil)

100 90

Non-rhizospheric soil Rhizospheric soil

80 70 60 50 40 30 20 10 0 15

30

45 Days

60

Figure 3.

(T-1) M. incognita (alone)

(T-2) M. incognita and D. dactyloides NDAd-05

(T-3) M. incognita and D. brochopagaNDDb-15

(T-4) M. incognita,+ D. dactyloides NDAd-05 + D. brochopaga NDDb-15

(T-5) M. incognita with carbofuran

(T-6) Control (untreated)

Figure 4.

(T-1) M. incognita (alone)

(T-2) M. incognita and D. dactyloides NDAd-05

(T-3) M. incognita and D. brochopagaNDDb-15

(T-4) M. incognita + D. dactyloides NDAd-05 + D. brochopagaNDDb-15

(T-5) M. incognita with carbofuran

(T-6) Control (untreated)

Figure 5.

(T-1) M. incognita challenged only

(T-2) M. incognita + D. dactyloides NDAd-05

(T-3) M. incognita + D. brochopaga NDDb-15

(T-4) M. incognita+ D. dactyloides NDAd-05+ D. brochopaga NDDb-15

(T-5) M. incognita + Carbofuran

(T-6) Control (untreated)

Figure 6.

(T-1) M. incognita challenged only

(T-2) M. incognita + D. dactyloides NDAd-05

(T-3) M. incognita + D. brochopaga NDDb-15

(T-4) M. incognita+ D. dactyloides NDAd-05+ D. brochopaga NDDb-15

(T-5) M. incognita + Carbofuran

(T-6) Control (untreated)

Figure 7. (a)

(e)

(b)

(f)

(c)

(g)

(d)

(h)

Table 1. Effect of D. dactyloides and D. brochopaga inoculation on H2O2 content, PAL, peroxidase, chitinase and SOD activity in the tomato leaves prechallenged with Meloidogyne incognita in pot experiments at 30 days of transplanting under nethouse condition. Treatments

H2O2 content (mmol

mg-1

PAL activity

protein) (Unit

g-1 fresh

Peroxidase activity wt.) (Unit

g-1 fresh

wt.)

Chitinase activity (Unit

g-1 fresh

wt.)

SOD activity (Unit g-1 fresh wt.)

T1- M. incognita

26.32 ± 1.33

10.33 ± 0.52

16.66 ± 0.70

13.34 ± 0.87

4.02 ± 0.45

T2- M. incognita + D. dactyloides NDAd-05

20.95 ± 1.05

15.87 ± 0.50

22.45 ± 0.56

19.75 ± 1.05

5.94 ± 0.41

T3- M. incognita + D. brochopaga NDDb-15

19.47 ± 0.75

15.50 ± 0.86

25.25 ± 0.95

20.55 ± 1.25

6.47 ± 0.25

T4- M. incognita + D. dactyloides NDAd-05 + D. brochopaga NDDb-15

12.25 ± 0.66

21.75 ± 0.98

32.47 ± 1.20

26.10 ± 1.50

9.25 ± 0.34

T5- M. incognita + carbofuran

9.45 ± 0.25

6.40 ± 0.25

10.50 ± 0.33

10.92 ± 0.67

2.46 ± 0.20

T6- Control (untreated)

5.26 ± 0.45

4.25 ± 0.22

7.49 ± 0.36

6.47 ± 0.25

1.75 ± 0.12

CD at 5%

1.75

1.96

2.25

1.25

0.50

Table 2. Effect of D. dactyloides and D. brochopaga inoculation on lignin and pectin content in the tomato roots pre-challenged with Meloidogyne incognita in pot experiments at 45 days of transplanting under nethouse condition.

Treatments

Lignin content (mg

g-1

dry wt.)

Uronic acid content (µg mg-1 dry wt.)

T1- M. incognita

16.47 ± 0.75

11.56 ± 0.47

T2- M. incognita + D. dactyloides NDAd-05

19.98 ± 1.10

13.75 ± 0.50

T3- M. incognita + D. brochopaga NDDb-15

20.23 ± 0.96

14.25 ± 0.34

T4- M. incognita + D. dactyloides NDAd-05 + D. brochopaga NDDb-15

24.33 ± 1.25

16.97 ± 0.25

T5- M. incognita + carbofuran

15.96 ± 0.56

10.54 ± 0.45

T6- Control (untreated)

14.25 ± 0.67

9.05 ± 0.30

CD at 5%

0.66

0.50

1 2 3 4

Table 3. Effect of D. dactyloides and D. brochopaga inoculation on root gall development and plant growth attributes in tomato pre-challenged with Meloidogyne incognita in pot experiments at 45 and 60 days of transplanting under nethouse condition. Treatm ents

Avg. number of galls/ seedling 45 60 DT DT 94.4 122. 1± 33± 2.03 5.93

Avg. number of nematode (J2)/ seedling

T2- M. incogni ta+ Arthro botrys dactylo ides NDAd05

62.2 5± 1.51

T3- M. incogni ta + Dactyl aria brocho paga NDDb15 T4- M. incogni ta+ Arthro botrys dactylo ides NDAd05 + Dactyl aria brocho paga NDDb15

T1Meloid ogyne incogni ta

Avg. shoot length (cm)

Avg. root length (cm)

Avg. fresh weight of shoot (g)

60 DT 16. 25± 1.4 5

45 DT 11. 20± 1.6 6

60 DT

45 DT

60 DT

18.27 ±1.24

7.33± 1.02

12.53 ± 1.75

Avg. fresh weight of root (g) 45 60 DT DT 8.3 11. 3± 60± 1.2 1.1 8 2

45 DT

60 DT

45 DT

2789.22 ±8.69

5562.50 ±11.25

12.27 ± 1.45

81.2 7± 2.04

1781.25 ±5.02

3252.75 ±8.50

28.30 ±2.23

46. 43± 1.5 2

22. 75± 1.2 5

36.03 ±1.30

25.24 ±2.33

32.40 ±1.66

4.9 0± 0.5 0

8.3 6± 1.2 5

69.6 6± 3.05

91.2 5± 1.51

1932.48 ±6.10

3596.24 ±9.29

26.25 ±2.15

42. 60± 1.6 5

19. 66± 1.6 6

32.10 ±1.40

21.75 ±2.05

30.25 ±2.05

5.3 9± 0.2 5

8.9 0± 0.5 0

47.2 5± 1.51

63.6 6± 1.04

1069.40 ± 4.05

1225.75 ±10.01

35.35 ±1.92

55. 66± 2.5 5

32. 48± 1.5 0

44.54 ±1.52

29.22 ±1.50

41.11 ±1.98

4.1 5± 0.1 1

6.8 5± 0.6 6

42

5

T5- M. incogni ta + Carbof uran

22.8 2± 1.06

30.9 2± 1.16

225.75± 3.80

596.75± 14.37

38.92 ±1.35

58. 90± 1.9 5

38. 33± 2.2 1

48.33 ±2.72

31.51 ±2.50

44.01 ±2.43

3.9 2± 0.1 5

6.2 4± 0.2 5

T6Control (untreat ed)

0.00 ±00

0.00 ±00

0.00±00

0.00±00

41.20 ±2.21

65. 22± 1.7 8

39. 13± 2.1 6

51.25 ±2.65

36.67 ±2.90

45.33 ±3.26

4.2 7± 0.5 0

6.5 0± 0.4 5

CD at 5%

4.96

6.50

45.96

36.85

2.15

2.3 3

1.7 5

2.50

2.10

1.96

0.4 5

0.3 3

Avg.- average, Control- untreated plants, DT- Days of transplanting, Data are mean ±SEM (n=5).

6 7

43

8 9 10

Highlights 1. Inoculation of D. dactyloides NDAd-05 and D. brochopaga NDDb-15 increased the antioxidant properties in the tomato

11

2. Stereoscopic visualization of H2O2 and superoxide radical in plant leaves was studied

12

3. D. dactyloides NDAd-05 and D. brochopaga NDDb-15 activated phenylpropanoid

13 14 15 16

pathway in tomato 4. Application of D. dactyloides NDAd-05 and D. brochopaga NDDb-15 increased cell wall lignification and pectin deposition 5. D. dactyloides and D. brochopaga mediated structural defense in tomato

17

44