Exometabolomic study of extracellular metabolites in tobacco plant induced by ethyl acetate extracts of Streptomyces diastatochromogenes KX852460

ABSTRACT Streptomyces diastatochromogenes KX852460, produced induce systemic resistance against Rhizoctonia solani AG-3. SA from the strain was given as treatment to the tobacco plants. Activity of the defense enzymes was analyzed by spectrophotometer, while extracellular metabolites were analyzed by Liquid Chromatography Tandem Mass spectrometer (LCMS-MS). Defense-related enzymes glutathione peroxidase (GR), glutathione peroxide (GPX), and peroxidase (POD) had maximum activity after treated with extract. Same as was observed in inoculated with R. solani, and extract induced the high activity of glutathione reductase (GST), catalase (CAT), phenylalanine ammonia-lyase (PAL), and polyphenol oxidase (PPO). While PPO had moderate activity even though with untreated samples (control). Metabolic profile of the treated sample was compared with control (untreated). Exometabolomic study reveal that in the treated sample amino acid (L-cysteine, N,S-di(trifluoroacetyl)-, trimethylsilyl ester), mammalian steroid 5α-Androstane-3α,17β-diol, bis(pentafluoropropionate), flavonoids (Rutin), amines and amides were identified. Current study determined that ethyl acetate extract could induce the resistance by activating the enzymes and by regulating the metabolism.


Introduction
Antagonistic microorganisms contribute a significant part in disease management of plants, by inducing the resistance in the plants, and unfolding the biological control mechanism (Ab Rahman, Singh, Pieterse, & Schenk, 2018). Tobacco plants have the ability to regulate the physiological and metabolic processes, by adjusting a sequence of mechanisms (Liu, Wang, Wang, Li, & Xin, 2014;Zhang, Zhang, Du, Chen, & Tang, 2011). Action of numerous enzymes (including superoxide dismutase, SOD; peroxidase, POD; catalase, CAT; glutathione-S-transferase, GST; glutathione reductase, GR; glutathione peroxidase, GPX; and non-enzymatic GSH; vitamin C, vitamin E, Ve; carotenoids; and antioxidants) alleviated the disturbed homeostatic mechanism of cells and stress damages (Anjum et al., 2015;Chen et al., 2016). Polyphenol oxidase (PPO) and proline oxidase (POX) have the role to build the defensive mechanisms by changing the composition of cell, and have the ability to catalyze the generation of oxidative phenols and lignin, to stimulate in contradiction to phytopathogens (Thilagavathi, Saravanakumar, Ragupathi, & Samiyappan, 2007). In the schematic biosynthesis of phenylpropanoid PAL enzyme have main role, and have key influence in the formation of flavonoid and in the biosynthesis of lignin (Podile & Laxmi, 1998). In the stress conditions, response mechanism of metabolites in tobacco leaf, allied with the perception of cure and period (Zhang et al., 2011). Phyto metabolomics in combination with the genomics, transcriptomics, and proteomics, analysis inclusive statics, for the broad spectrum determination of plant growing, enlargement, resistance and production (Misra, Assmann, & Chen, 2014). Several bacterial metabolites besides phyto hormones have been investigated (Amin et al., 2015;Johnson, Kido Soule, & Kujawinski, 2016;Paerl et al., 2017). Recently, many researchers investigated the plants and microbes exomatabolomic study (Jacoby, Martyn, & Kopriva, 2018: Wienhausen, Noriega-Ortega, Niggemann, Dittmar, & Simon, 2017. Metabolic responses of tobacco to the environment have been vastly investigated, however, comparatively few works have been done to analyze the metabolic changes induced by microbial extract in tobacco plants. In our previous study, we isolated, identified, and characterized Streptomyces diastatochromogenes KX852460 strain for the biological control of tobacco target spot. (Ahsan, Chen, Wu, Irfan, & Shafi, 2017;Ahsan, Chen, Zhao, Irfan, & Wu, 2017;. The aim of the present study is to induce the systemic resistance in tobacco against Rhizoctonia solani AG-3. . Given the name to the ethyl acetate extract of S. diastatochromogenes KX852460 as SA.

Enzyme extraction
Treatments were given in replica 24-h pre-inoculation of R. solani AG-3 and 24-h post inoculation of R. solani. Plants were inoculated and treated at the same time. Treatment with distilled water was considered as control. Plants were kept in hygienic conditions at 28°C. Selected the leave randomly from different plants to make the homogenate for the analysis. For total enzyme extracts, 0.2 g of callus were ground to fine powder with liquid nitrogen and homogenized with a mortar and pestle in 2 ml of ice cold 50 mM Tris-HCl buffer (pH 6.8) containing 0.1 mM ethylene-diaminetetraacetic acid (EDTA), 5 mM cysteine and 1% PVP. The homogenate was centrifuged at 8000 g for 15 min, and the supernatant fraction was filtered through a column containing 1 ml of Sephadex G-50 equilibrated with the same buffer was used for homogenization. All operations were performed at 4°C. The filtered fraction was used for enzyme activity (Zhao et al., 2012).

Determination of enzyme activities
GST, GR, and GPX activity was assayed spectrophotometrically as described by the method of (Drotar, Phelps, & Fall, 1985). POD activity was determined following the method described by (Yao & Tian, 2005), CAT, SOD, phenylalanine ammonia-lyase (PAL) and PPO by the method of (Li & Steffens, 2002).

Analysis of metabolites changes in tobacco plant induced by the SA
Plant treated with SA and plant without treatment was considered as control (CK). After 36 h, leaf sample was taken and ground in different fractions of methanol followed by centrifugation at 10,000 rpm for 15 min. Mass spectrometer operating in electrospray ionization (ESI) negative mode and hyphenated with an Agilent 1290 ultra-high performance liquid chromatography system was used. The high purity nitrogen gas for the mass spectrometer was set at 30 psi for source gas, 30 psi for the heating gas and high for collision gas with a source temperature of 500°C. The setting for ESI voltage was set to 4500 kV. The collision energy to attain fragmentation was set at 35 eV with a spread of ±15 eV.
Mass range for MS/MS scan was set from 50 to 1000 m/z while mass range for full scan was set from 50-1000 m/z while scan speed was set at 1000 m/z per second. A DIKMA Leapsil C18 (150 mm × 4.6 mm i.d. 2.7 µm particle size was used to obtain separation. The mobile phase was made up of aqueous ammonium formate (5 mmol/L) with 0.1% formic acid (solvent A) and acetonitrile with ammonium formate (5 mmol/L) with 0.1% formic acid (solvent B). The compounds were separated with the following linear-programmed solvent gradient: 0 min (10% B), 10 min (95% B), 2 min (95% B) then equilibrating back to 10% B for 3 min. The flow rate for the column was set at 0.4 ml/min while the column temperature was set at 30°C and injection volume at 20 µL. Data were matched with NIST chemistry Web Book.

Statistical analysis
All the data were statistically analyzed using Minitab software version 17. The data presented were the mean of triplicates.

Activity of GST in tobacco leaves of different treatments
When the SA sprayed over the leaves of tobacco plants, activity of GST were increased up to 4 days and then decreased in untreated (Ck) samples. When tobacco leaves were treated with culture filtrate of Streptomyces sp. (T), the enzyme activity was at its peak on 6 th day and then declined up to 14 th day. When tobacco leaves were inoculated with R. solani AG-3 (Y) the enzyme showed similar pattern like untreated samples. Tobacco leaves treated with both culture filtrate and R. solani AG-3 (TY), the GST activity become highest at 2 nd day and then reduced with increased time as shown in Figure 1.

Activity of GR in tobacco leaves of different treatments
Activity of GR was increased up to 4 days and then become decreased in untreated (Ck) samples. When tobacco leaves were treated with SA (T) the enzyme activity was at its peak on 6 th and 12 th days and then declined up to 14 th day. When tobacco leaves were inoculated with R. solani AG-3 (Y) the enzyme activity increase on 12 th day and then declined on 14 th day. Tobacco leaves treated with both SA and inoculated by R. solani AG-3 (TY), the GR activity become highest at 10 th and 12 th days and then reduced with increased time as shown in Figure 2.

Activities of GPX in tobacco leaves of different treatments
Activity of GPX was increased on 2 nd , 4 th , 6 th , 8 th , and 12 th days, while the activity of GPX decreased on 10 th and 14 th day in untreated (Ck) samples. When tobacco leaves were treated with SA (T) the enzyme activity was at its peak on 2 nd day and then declined up to 14 th days except on the 8 th day there was slightly increased in activity. When tobacco leaves were inoculated with R. solani AG-3 (Y), the enzyme activity increased on 2 nd , 4 th , and 8 th day, while on 6 th , 10 th , 12 th , and 14 th day decreased. Tobacco leaves treated with both SA and R. solani AG-3 (TY), the GPX activity was highest at 0, and 6 th day, while on 14 th days the activity was maxima. Whereas decline in GPX activity was observed on 2 nd , 8 th , 10 th , and 12 th day as shown in Figure 3. 3.4. Activity of POD in tobacco leaves of different treatments POD activity was increased on 2 nd , 4 th , and 6 th day and then decreased up to 12 th day, while on the 14 th day again activity increased in untreated (Ck) samples. When tobacco leaves were treated with SA (T), the enzyme activity was at its peak on 4 th , 6 th , 10 th , and 12 th day, whereas declined the activity on 0, 2 nd , 8 th , and 14 th day. When tobacco leaves were inoculated with R. solani AG-3 (Y), the enzyme showed similar pattern like untreated samples. Tobacco leaves treated with SA and R. solani AG-3 (TY), the GST activity was high from 2 nd to 10 th day and then reduced with increased time as shown in Figure 4.

Activity of CAT in tobacco leaves of different treatments
Activity of CAT was increased on 2 nd and 4 th day and then decreased in untreated (Ck) samples. When tobacco leaves were treated with SA (T), the enzyme activity was at its peak on 6 th day and then declined up to 14 th day. When tobacco leaves were inoculated with R. solani AG-3 (Y), the enzyme showed good activity on 2 nd and 4 th day, while afterwards decreased the activity up to 14 th day. Tobacco leaves treated with both SA and R. solani AG-3 (TY), the GST activity was high at 2 nd day and then reduced with increase in time as shown in Figure 5.

Activity of SOD in tobacco leaves of different treatments
Activity of SOD was increased on 12 th day and then decreased in untreated (Ck) samples. When tobacco leaves were treated with SA (T), the enzyme activity was at its peak on 6 th day and then declined up to 14 th day. When tobacco leaves were inoculated with R. solani AG-3 (Y), activity of SOD on 8 th and 12 th day increased. Tobacco leaves treated with both SA and R. solani AG-3 (TY), the SOD activity was observed high at 10 th and 14 th day increased as shown in Figure 6.

Activity of PAL in tobacco leaves of different treatments
Activity of PAL was increased up to 6 days and then decreased in untreated (Ck) samples. When tobacco leaves were treated with SA (T), the enzyme activity was at its peak on 4 th to 6 th day, and as well again increased on 10 th and 12 th day, whereas declined up to 14 th day. When tobacco leaves were inoculated with R. solani AG-3 (Y), the enzyme showed overall good activity, but high activity was observed on 8 th day. Tobacco leaves treated with both SA and R. solani AG-3 (TY), the PAL activity become high at 2 nd to 10 th day and then reduced with increased time as shown in Figure 7.

Activity of PPO in tobacco leaves of different treatments
Activity of PPO was increased on 4 th and 12 th day, while throughout all other days decreased in untreated (Ck) samples. When tobacco leaves were treated with SA (T) the enzyme activity was at its peak up to 2 days and then declined up to 14 th day. When tobacco leaves were inoculated with R. solani AG-3 (Y), the activity of PPO was increased on 0 day. Tobacco leaves treated with both SA and R. solani AG-3 (TY), the PPO activity was high at 0 to 2 nd day and again raised on 14 th day, whereas activity decreased with increased time as shown in Figure 8.

Analysis of metabolites changes in tobacco plant induced by the SA
Exometabolomic study was carried out for the identification and quantification of metabolic changes in tobacco leave by treating with SA. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) was used to analyze the profile of metabolites in treated leave and non-treated leave (control). Mass spectrum of the identified extracellular metabolites is given in Figure 9. In the treated sample amino acids, organic acids and alkanes, derivatives, amides and their derivatives, derivatives of amines, mammalian steroid, and flavonoids were determined. Long chain fatty acid such as Oleic acid, 2,3-bis-(OTMS) propyl ester was found. The most important was the presence of 5α-Androstane-3α, 17β-diol, bis (pentafluoropropionate) steroid in the treated sample. As by comparison to the non-treated sample (Ck), the amino acids were identified in higher quantity. L-Cystathionine,N,Nbis(tert-butyldimethylsilyl)-,bis(tert butyldimethylsilyl) ester, Sebacic acid, di(2,2,3,3,4,4,5,5-octafluoropentyl) ester, and L-Methionine, N-heptafluorobutyryl-, heptadecyl ester, organic acids and alkanes were found in nontreated sample. None of the steroids, flavonoids, amines, and amides was determined in the control (Table 1) Methionine, N-heptafluorobutyryl-, heptadecyl ester and L-Cystathionine, N, N-bis(tert-butyldimethylsilyl)-,bis (tert-butyldimethylsilyl)ester has been occurred. As these amino acids were found in control sample, which was extracted from the same growing conditions plants of tobacco and these amino acids, are the precursor of cysteine and derivatives of cysteine.

Discussion
To considering this point of view to induced the systemic resistance in tobacco against tobacco target spot. A systemic analysis of enzymes and metabolism was conducted. In the current study, eight kinds of defense enzymes were analyzed: GST, GR, GPX, POD, CAT, SOD, PAL and PPO. Defense-related enzymes GR, GPX, and POD had more activity after treated with secondary metabolites. Whereas GST, CAT, PAL, and PPO had more efficiency treated with secondary metabolites spray and inoculation at the same time. While PPO had moderate activity even though with untreated samples (control), indicated that PPO activity level was near the same to the each other.
Activation of these defense enzymes demonstrated, that defensive enzymes burst the ROS and protect the plant cells from the oxidative damage. It was observed that the tobacco plants showed restricted adaptation against the R. solani AG-3 inoculation. Induction of GST by the ROS due to the oxidative stress, metabolomic regulation of toxic substances, and former molecules was done by GST enzyme (Liu et al., 2013). GR also has a key role to poise the redox phenomenon in the cell (Mohan et al., 2015). Reactive oxygen species (ROS) caused oxidative rupture, so the plants adopt intricate defensive pathway to foraging the ROS. This mechanism comprises of enzymatic scavenging systems, likewise POD, CAT, and SOD (Howe & Schilmiller, 2002). Catabolism of   biosynthetic suberin and lignin can be by POD (Ingham, Parker, & Waldron, 1998). Homeostatic mechanism of PPO has a variety of phenomena, and stimulates the reaction to respond the mechanical burst, invasion of fungus and bacteria, physiological stresses, and signs to the jasmonic acid, SA, Ethylene and CAMP (Thipyapong & Steffens, 1997). PAL has significant role in the metabolic process of phenyl propanoid and causes the production of phenols (Massala, Legrand, & Fritig, 1980). Ethyl acetate extracts of S. diastatochromogenes KX852460 showed on tobacco leaf. Exometabolomic study of tobacco leaf by the LC-MS/MS indicated that metabolic flux of treated sample with ethyl extract was different than control (Ck). In the treated sample, there were two amino acids L-Cysteine, N,S-di(trifluoroacetyl)-, trimethylsilyl ester and D-Alanine, N-(4ethyl benzoyl)-, tridecyl ester were determined in the MS/MS analysis. As these amino acids were found in control sample, which was extracted from the same growing conditions plants of tobacco and these amino acids, are the precursor of cysteine and derivatives of cysteine. Important biomolecules such as vitamins, cofactors, antioxidants, and many defense compounds originated from the metabolism of cysteine (Droux, 2004;Wirtz & Droux, 2005).
Homeostasis of cysteine has been acting as a vital protagonist in the plant immunity (Álvarez, Ángeles, Romero, Gotor, & García, 2012). Organic acids and alkane were identified in both samples. As the organic acids have essential role in various plant metabolic pathways (Ludwig, 2016). Higher alkanes have the inhibitory effects against the microbial pathogens. Presence of long chain fatty acid derivatives (Oleic acid, 2,3-bis-(OTMS) propyl ester) in the treated sample indicated that ethyl acetate extract induced the immunity in the tobacco plant. Biosynthesis pathway of long chain fatty acids belongs to the resistance of the plant against the pathogens (Raffaele, Leger, & Roby, 2009). Presence of 5α-Androstane-3α,17β-diol, bis(pentafluoropropionate) steroids in the treated sample is the key point.
According to recent reports, derivatives of androstane are endogenous antagonist of constitutive receptor in human, and CAR is a sensor of exobiotic and endobiotic (Wada, Gao, & Xie, 2009). Whereas some researchers reveal that mammalian steroids have effects on the plant growth, cell divisions, root and shoot growth, embryo growth, flowering, and pollen tube growth and callus proliferation (Lindemann, 2015). Rutin, a flavonoid, was detected in the treated sample, and the flavonoids are the groups of secondary metabolites, which are mostly exist in the leaves of the plants. These flavonoids not only building blocks of the plants, but also has the immunity against the pathogens (Galeotti, Barile, Curir, Dolci, & Lanzotti, 2008). Derivatives of amines and amides were also identified in the treated sample. Amines and amides are the secondary metabolites and plant secondary metabolites are antimicrobial. Results of MS/MS spectrum of the control sample showed only amino acids, organic acids, and alkanes. None of the fatty acid, flavonoid, amines, and amides was identified in the non-treated sample. By comparison to the treated sample, the profile of non-treated sample was basic compounds amino acids and alkanes, which could induce the systemic resistance in the tobacco (Sharma, Vineet, Dubey & Maheshwar, 2018) Exometabolomic study revealed that the treatment of ethyl acetate extract of S. diastatochromogenes KX852460 induced the inhibitory metabolites in the tobacco plants. Several studies indicated that in plant diversity, bacterial microbes can induce the defense substances for the soil borne disease management (Nagórska, Bikowski, & Obuchowski, 2007). Current study demonstrated that ethyl acetate extract of S. diastatochromogenes KX852460 can induce the defense response in tobacco plant.

Conclusion
Ethyl acetate extracts of S. diastatochromogenes KX852460 induced the resistance by activate the defense enzymes. Defense-related enzymes GR, GPX, and POD had more activity after treated with microbial extract. Whereas GST, CAT, PAL, and PPO had more efficiency treated with secondary metabolites spray and inoculation at the same time. While PPO had moderate activity even though with untreated samples (control), indicated that PPO activity level was near the same to each other. Activation of these defense enzymes demonstrated that defensive enzymes showed restricted adaptation against the R. solani AG-3 inoculation. Exometabolomic study of plant leaf indicated after treated the extracts of ethyl acetate that metabolism changes have been occurred in tobacco leaves and flux of the metabolites were changed. Amino acid (L-Cysteine, N,S-di(trifluoroacetyl)-, trimethylsilyl ester), mammalian steroid 5α-Androstane-3α,17β-diol, bis(pentafluoropropionate), flavonoids (Rutin), amines, and amides were identified. The current study determined that ethyl acetate extract of Streptomyces diastatochromogenes KX852460 activated the defense enzymes. Modulation in metabolic profile of tobacco could produce immune response against the pathogen.

Disclosure statement
No potential conflict of interest was reported by the authors.