New insights on the utilization of ultrasonicated mustard seed cake: chemical composition and antagonistic potential for root-knot nematode, Meloidogyne javanica

New insights on the utilization of ultrasonicated mustard seed cake: chemical composition and antagonistic potential for root-knot nematode, Meloidogyne javanica 利用超声波芥菜籽饼的新见解：化学成分和对根结线虫的拮抗潜力 1 first-author 2 1 3 1 4 1 corresp Faheem AHMAD, ahmad_nematol@yahoo.com Faheem AHMAD, ahmad_nematol@yahoo.com ahmad_nematol@yahoo.com https://orcid.org/0000-0002-7450-0900 Department of Botany, Aligarh Muslim University, Aligarh-202002, U.P., India Department of Botany, Aligarh Muslim University, Aligarh-202002, U.P., India Department of Botany, School of Biological Sciences, North-West University (Mafikeng Campus), Mmabatho 2735, South Africa Department of Botany, School of Biological Sciences, North-West University (Mafikeng Campus), Mmabatho 2735, South Africa Pharmacopoeia Commission for Indian Medicine and Homeopathy, Ghaziabad-201002, U.P., India Pharmacopoeia Commission for Indian Medicine and Homeopathy, Ghaziabad-201002, U.P., India Faculty of Agriculture and Natural Resources, University of Namibia, Windhoek10026, Namibia Faculty of Agriculture and Natural Resources, University of Namibia, Windhoek10026, Namibia This study focused, for the first time, on the effect of ultrasonic features on the extraction efficiency of secondary metabolites in mustard seed cake (MSC). The nematostatic potential of sonicated seed cake was examined against the second-stage juveniles (J2s) of root-knot nematode, Meloidogyne javanica . The results show that a 35 ppm (parts per million) concentration of a sonicated extract (SE) sample of MSC caused 65% J2s mortality at 18 h exposure period in vitro. It also significantly suppressed the root-knot index (RKI=0.94) in tomato roots. The lethal concentration values for SE were 51.76, 29.79, and 13.34 ppm, respectively, at 6, 12, and 18 h of the exposure period, and the lethal concentration values for the non-sonicated extract (NSE) sample were 116.95, 76.38, and 55.59 ppm, respectively, at similar exposure time. Sinapine and gluconapin were identified as the major compounds in ultrasonic-assisted MSC. Because of the high extraction efficiency of metabolites in the SE, all treatments of SE were shown to be antagonistic to J2s. Thus, this study of ultrasonication activity-based profiling of MSC may help generate target-based compounds at a scale relevant to the control of disease caused by nematodes in economic crops. 目的 2 研究超声芥菜籽饼（MSC）对次生代谢产物提取效率的影响。 创新点 2 基于MSC超声活性分析的研究有助于规模生产经济作物中与控制线虫引起的疾病有关的目标化合物。 方法 2 在玻璃烧杯中用100 mL溶剂混合物制备MSC粉末（10 g），然后放入数字超声波浴中以获得超声提取物（SE）滤液，或将MSC混合物在带有磁力搅拌器的锥形烧瓶中搅拌以获得非超声提取物（NSE）滤液。这些获得的滤液用于扫描电子显微镜（SEM）成像、液相色谱电喷雾电离质谱（LC-ESI-MS）系统分析化学成分并评估对根结线虫的拮抗潜力。 结论 2 超声处理过的MSC对根结线虫（Java）的二期幼虫（J2s）具有抑瘤潜力，且显著抑制了番茄根的根结指数（RKI），值为0.94。在相似的暴露时间下，SE的致死浓度（LC 50 ）值分别在6、12和18小时分别为51.76、29.79和13.34 ppm（10 −6 ），NSE的LC 50 值分别为116.95、76.38和55.59 ppm。在MSC的SE中，芥子酸胆碱和葡萄糖芫菁芥素被确定为主要化合物。由于SE中代谢物的高提取效率，所有SE均显示出对J2s的拮抗作用。研究结果表明，在体外暴露18小时，浓度为35 ppm的MSC的SE样品引起了65％的J2s死亡率。 Plant-parasitic nematode Plant by-product Sono-extraction Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) Natural compound Nematode management 植物寄生线虫 植物副产品 声音提取 液相色谱电喷雾电离质谱（LC-ESI-MS） 天然化合物 线虫管理 1 Introduction 1 The Meloidogyne genus (root-knot nematode) has a worldwide distribution and is the cause of severe crop losses worldwide ( Wesemael et al., 2011 Wesemael et al., 2011 ). Of 70 known Meloidogyne spp., only Meloidogyne javanica , Meloidogyne incognita , Meloidogyne arenaria , and Meloidogyne hapla are of major economic importance. The severity of damage caused by Meloidogyne spp. is often species-specific and varies depending on plant species, crop rotation, season, and soil type ( Potter and Olthof, 1993 Potter and Olthof, 1993 ). The key and most common symptom of infected plants is the appearance of root galls within their root systems, in which the Meloidogyne spp. are embedded. The infected host plants are thus severely affected, and the plants become susceptible to wilting, growth reduction, and infection by other plant pathogens. Effective management methods to combat root-knot nematodes include synthetic nematicides, which is the preferred method for farmers in most countries ( Whitehead, 1997 Whitehead, 1997 ). However, these synthetic nematicides harm our natural environment and are especially harmful to non-target organisms of the host plants. In recent years, a new environmentally friendly direction based on plant by-products has been developed as an option to achieve sustainability in agriculture ( Rahman and Somers, 2005 Rahman and Somers, 2005 ; Valdes et al., 2012 Valdes et al., 2012 ). Advances in sustainability science, including the recent development of coupled plant by-product sono-extraction, provide a new context for integrating knowledge about compound extraction. In addition, the selection of plant species and their by-products is crucial. An improperly selected plant species/by-product reduces or harms populations of beneficial soil microorganisms that prey on plant-parasitic nematodes ( Henderson et al., 2009 Henderson et al., 2009 ). In this context, identifying plant species and their by-products that act as antinematodal agents and the elucidating of their mechanism of action might serve the purpose of controlling nematode infection. Brassicaceous crops and their by-products, such as seed cake, although often utilized for nematode control, host a wide range of plant-parasitic nematodes, including Meloidogyne spp. ( Rahman and Somers, 2005 Rahman and Somers, 2005 ; Valdes et al., 2012 Valdes et al . , 2012 ; Fourie et al., 2016 Fourie et al . , 2016 ). The plant by-products resulting from oil extraction are largely defatted seed cakes, with 32.02 Mt produced in 2019 in the European Union alone ( FEDIOL, 2020 FEDIOL, 2020 ). Thus, its disposal and its evaluation as a by-product are a global challenge. Recently, oil cakes have become an attractive source for producing bioactive substances such as phenolic and flavonoid compounds ( Ramachandran et al., 2007 Ramachandran et al . , 2007 ; Teh and Birch, 2014 Teh and Birch, 2014 ). Similarly, the biocidal compounds released from the seed cake of Brassica spp. were found to be toxic to soil-borne diseases and pests ( Rahman and Somers, 2005 Rahman and Somers, 2005 ; de Corato et al., 2015 de Corato et al . , 2015 ). Secondary metabolites of seed cake are enclosed in rigid and insoluble structures such as vacuoles, lipoprotein bilayers, lignin, hull, and cell walls ( Corrales et al., 2008 Corrales et al . , 2008 ). So the choice of extraction method is critical. For sufficient adoption, techniques and tools are needed to resolve research challenges for filling gaps regarding promising secondary metabolites for nematode management strategies. Our research interest has concentrated on increasing the extraction efficiency for secondary metabolites of mustard seed cake (MSC) for further use. Ultrasonication is one of several rapidly emerging techniques devised to enhance quality and processing, minimize the use of organic solvents, and maximize the bioavailability of organic products ( Knorr et al., 2011 Knorr et al . , 2011 ; Rincón et al., 2013 Rincón et al . , 2013 ). The representative range for the frequency that is generally used in these ultrasonic techniques lies between 20 kHz and 500 MHz ( Yusaf and Al-Juboori, 2014 Yusaf and Al-Juboori, 2014 ). During the extraction process, a considerable part of the energy of the ultrasonic waves converts into thermal energy. Ultrasonic waves pass through a conductive medium while putting pressure on the medium. Ultrasound ranging from 20 to 100 kHz is used in chemically critical systems in which chemical and physical changes are required, as it can cause cavitation ( Pilli et al., 2011 Pilli et al . , 2011 ). A significant advantage of this technique over other tools that are used to characterize materials is that it is a non-invasive, non-destructive, economic, eco-friendly, and sustainable technology and can operate in concentrated and solid materials ( Teh and Birch, 2014 Teh and Birch, 2014 ). Potential applications of ultrasonic techniques have been reported and discussed to understand changes of content as well as extraction capacity of secondary metabolites, in particular phenolics, polyphenol content, and antioxidants within the extracted samples of various seed cakes undergoing ultrasonic treatment ( Teh and Birch, 2014 Teh and Birch, 2014 ; Khalili and Dinani, 2018 Khalili and Dinani, 2018 ). When ultrasonic treatment is applied to a liquid, ultrasonic waves are tied with a cyclic succession of expansion (rarefaction) and compression phases imparted by mechanical vibration ( Tang, 2003 Tang, 2003 ). This technique has high efficiency and low extraction time in accelerating promising natural compounds ( Picó, 2013 Picó, 2013 ). In this study, experiments were conducted to evaluate the nematostatic potential of ultrasonic-assisted MSC against the second-stage juveniles (J2s) of M. javanica and to assess the potential strength of sonicated extract (SE) samples when used in various experimental strategies. In addition, we characterized the composition of secondary metabolites, including nematostatic compounds, in the SE samples by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). We also investigated the morphological variation of ultrasonic-assisted MSC by scanning electron microscopy (SEM). The current method can be applied as a sustainable approach to enhancing secondary metabolite extraction efficiency in MSC extract. 2 Materials and methods 1 2.1 Materials and reagents 2 Defatted cake of Brassica campestris L. (mustard) was obtained from the Oil Seed Extractions Mill of Aligarh, India. Air-dried seed cake was ground with a blender (Black & Decker BXBL6001IN, USA) to produce the powder. The dried powder was then passed through a 300-mesh-size sieve, placed in a plastic bag, and stored in an airtight container at 4 ℃ prior to analysis. Methanol and acetone of analytical grade were purchased from Sisco Research Laboratories (SRL) Pvt. Ltd., India. Ultra-pure water was obtained from the Thermo Scientific Barnstead Ultrapure Water System (Gen Pure Pro UV-TOC/UF, Sweden). 2.2 Ultrasonic bath treatment of mustard seed cake 2 MSC powder (10 g) was prepared with 100 mL of the solvent mixture in a glass beaker. The solvent mixture composition was prepared with methanol, acetone, and water in a 7:7:6 volume ratio and then loaded in a digital ultrasonic bath (Labman Scientific Instruments Pvt. Ltd., India. The digital ultrasonic bath was set at 40 kHz and 100 W of power at room temperature for 35 min. The filtrates were then placed in a centrifuge at 3000 r/min for 15 min, and the supernatant was collected in an amber reagent bottle before sample preparation for analysis. This sample was termed sonicated extract (SE). 2.3 Non-ultrasonic bath treatment of mustard seed cake 2 MSC (10 g) prepared in 100 mL of mixed solvent (methanol, acetone, water in a 7:‍7:‍6 volume ratio). The mixture was stirred in a conical flask with a magnetic stirrer at room temperature for 35 min. Filtrates were then placed in the centrifuge at 3000 r/min for 15 min, and the supernatant was collected in an amber reagent bottle before sample preparation for analysis. This sample was termed non-sonicated extract (NSE). 2.4 SEM of sonicated extract 2 The morphological alterations in the extracted samples were analyzed using SEM (JSM 6510 LV, Jeol, Tokyo, Japan). Dry filtrates were obtained by heating with a burner for approximately 1 min, and the precipitates were scratched out in a 2-mL Eppendorf tube for SEM analysis. The dried filtrates were carefully placed on a clean aluminium stub which had double-sided tape and was gold-coated (JEOL JFC-1600 fine auto coater) with a layer about 14 nm thick. After this, the aluminium stubs were loaded into the SEM for imaging. 2.5 Sample preparation 2 The collected supernatants of both SE and NSE were further filtered separately via a syringe filter of 0.22 μm diameter (Millipore, Germany). The filtrates were diluted with methanol to the final working concentration for analysis. The sample solutions were stored at -20 ℃ until use and vortexed before injection into the LC-ESI-MS system for analysis. A mere 0.50-μL aliquot was found to be sufficient for injecting into the LC-ESI-MS system. 2.6 LC-MS analysis 2 The analysis of the MSC extract was performed on an accelerated ultra-performance liquid chromatography (UPLC) system equipped with a pump, autosampler, and photodiode array (PDA) detector. A tandem quadrupole detector (TQD) triple quadrupole ESI ion trap mass spectrometer was coupled to the LC system (Waters, Milford, MA, USA). The UPLC column used was an Accucore C18 column (150 mm×2.1 mm, 2.6 µm). Dual-mode (±) LC-ESI-MS experiments were performed after injecting 0.50 µL extract with the autosampler. Water acidified with 0.1% (volume fraction) formic acid (Eluent A) and acetonitrile acidified with 0.1% (volume fraction) formic acid (Eluent B) were used as solvent at a constant flow rate of 0.250 µL/min. The column temperature was maintained at 30 ℃. The elution gradient used was: 0 min (5% B), 0‒1 min (5% B), 1‒6 min (5% to 30% B), 6‒12 min (30% to 60% B), 12‒16 min (60% B), 16‒20 min (60% to 80% B), 20‒24 min (80% B), 24‒26 min (80% to 5% B), and 26‒30 min (5% B). MS analysis was performed on a TQD triple quadrupole equipped with an ESI-MS probe coupled to UPLC. Mass spectra were recorded in a mass-to-charge ratio ( m / z ) range of 150‍‒‍2000 Da, and data were acquired and processed using the MassLynx TM software (Waters Corporation). Accurate masses of selected metabolites were observed and compared with their exact mass (theoretical mass) values manually. The m / z values of identified compounds with retention time are listed in Tables 1 1 and 2 2 . RT: retention time; ACN: acetonitrile; PND: peak not determined; SE: sonicated extract; NSE: non-sonicated extract; m / z : mass-to-charge ratio, considered as the mass; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry. 2 RT (min) 2 Secondary nematostatic compound 2 Molecular ion mass ( m / z ) 2 Adduct molecular ion mass ( m / z ) 2 Area (%) SE NSE 1.14 3-Methylsulfinyl propyl isothiocyanate 163.01 6.2 6.0 4.76 Sinapine 310.16 34.5 26.3 5.71 1-Methoxybrassitin 250.07 496.2 [M+2H] + 3.6 PND 6.77 4-Hydroxybenzyl glucosinolate 425.04 458.2 [M+CH 3 OH+H] + 2.2 0.9 7.86 Patuletin 3-gentiobioside 656.15 8.2 6.5 8.03 Isorhamnetin 3,7-di- O -β-glucopyranoside 640.16 682.3 [M+ACN+H] + 13 PND 11.44 Rutalexin 232.03 274.3 [M+ACN+H] + 7.8 5.3 14.20 Benzeneacetaldehyde 120.05 162.2 [M+ACN+H] + 2.2 2.0 14.72 5-Methylthiopentylglucosinolate 435.06 477.1 [M+ACN+H] + 8.4 6.3 17.10 Campesterol 400.37 2.8 2.8 18.50 Coumestrol 268.03 301.2 [M+CH 3 OH+H] + 3.7 2.0 22.22 Methyl oleate 296.27 338.3 [M+ACN+H] + 2.6 2.5 26.07 4-Hydroxyglucobrassicin 464.05 885.5 [M+ACN+2H] + 4.8 2.3 ACN: acetonitrile; PND: peak not determined; RT: retention time; SE: sonicated extract; NSE: non-sonicated extract; m / z : mass-to-charge ratio, considered as the mass; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry. 2 RT (min) 2 Secondary nematostatic compound 2 Molecular ion mass ( m / z ) 2 Adduct molecular ion mass ( m / z ) 2 Area (%) SE NSE 1.47 Gluconapin 373.05 372.1 [M‍-‍H] - 32.2 25.4 6.17 Se-Methylselenocysteine 182.97 545.1 [3M‍-‍H] - 8.2 PND 14.12 Methoxybrassinin 266.05 265.2 [M‍-‍H] - 16.5 12.3 19.92 Indole-3-acetonitrile 156.06 311.1 [2M‍-‍H] - 2.0 1.8 2.7 Isolation, culture amplification, and identification of M. javanica 2 The M. javanica (root-knot nematode isolate) inoculum was produced on susceptible eggplant plants. Nematode egg masses were extracted from the infected roots. The egg masses were then placed on a sieve (8.5 cm in diameter), which was already layered with tissue papers over glass Petri plates (10 cm in diameter) at 25 ℃ to obtain J2s as inoculum. J2s were collected from the bottom of the Petri plate, and those that emerged from 2 to 5 d were stored at 9 ℃. Aliquots of the J2s suspensions were placed in growth chambers for 8 h before inoculation to acclimatize J2s to the testing temperatures. SEM was used for species identification. An adult female of M. javanica was dissected from the infected eggplant root, and the perineal pattern was prepared according to the technique previously described by de O. Abrantes and de A. Santos (1989). The adult female was placed in a few drops of lactic acid (45%) on a transparent glass slide. The posterior portion with cuticular markings surrounding the vulva and anus of the female was cut using a sharp blade to obtain the perineal pattern. The cut section was then transferred onto another glass slide, and a drop of lactic acid (45%, volume fraction) was placed on a round coverslip, which was rimmed with a thick ring of glycerol. A few drops of formalin (2%, volume fraction) were then added to the glycerol every 2‒3 min to wash out the lactic acid. The perineal patterns were allowed to dry in a glass desiccator at room temperature. Before SEM analysis, the coverslip was detached from the glass slide, mounted onto an SEM stub with double-sided adhesive tape, and coated with 14 nm of gold. It was then examined in the SEM (JSM 6510 LV, Jeol, Tokyo, Japan). The surface morphology of the perineal pattern of M. javanica was reviewed from the SEM images ( Fig. 1 Fig. 1 ). The key features of the perineal pattern were observed. These were the presence of a low dorsal arch, smooth striae, a tail whorl often distinct, a distinct lateral field, and demarcation from the striae by more or less parallel lines. 2.8 J2s mortality and J2s mobility recover tests 2 For the mortality test, 200 J2s were incubated in small Petri plates of 5 cm in diameter, containing 2 mL of SE and NSE samples extracted separately. The mortality test was exposed to varying concentrations of extracted samples, namely, 5, 15, 25, and 35 ppm (parts per million). J2s kept in Petri plates filled with only 2 mL distilled water (DW) were considered as control. J2s were washed three times with water and re-suspended in a counting dish, and both mobile and dead J2s were recorded at a 6, 12, and 18 h interval from the incubation period. J2s without detectable movement were considered dead. The mortality test was performed with four replicates. Subsequently, a J2s mobility recovery test was also performed. The recovery strength of J2s was analyzed after 18 h of incubation in 35 ppm of SE samples. To confirm recovery strength, J2s were washed three times and re-suspended in water to check for movement. Mobile and immobile J2s were counted at 1, 3, 5, and 7 d to examine their capability to recover mobility. Maximum immobility (65%) was observed on the first day after incubation, but a significant recovery in J2s mobility was also found at 3 ‍d. Furthermore, the percentage of immobile J2s (40%) at 5 d after 18 h of incubation in the SE sample (35 ppm) was not significantly different from that in a similar suspension at 7 d, indicating that J2s cannot recover further. These data collectively indicate that an SE solution (35 ppm) has a toxic effect on J2s ( M. javanica ) mobility. 2.9 Root-knot index assessment-pot study 2 Bare root-dip treated tomato seedlings (two weeks old) in a 35-ppm extract of MSC were transplanted into 15-cm-diameter clay pots filled with 1.5 kg autoclaved soil mixtures (sand and organic matter). Before transplanting, part of the root was dipped in 35 ppm of SE and NSE separately for a 30-min duration. Three days after the transplantation, about 2500 J2s of M. javanica were root inoculated into each tomato plant. The tomato seedlings without J2s treatment were considered as control. Post-transplantation, each tomato seedling was irrigated with water regularly to maintain moisture in the rhizospheric soil. Four replicates were set up for each treatment. Sixty days later, the pot experiment was terminated for assessment of root-knot index (RKI) severity in the tomatoes. The RKI was calculated using the standard scale method of 0 to 5, where 0=no galls, 1=1 to 2 galls, 2=3 to 10 galls, 3=11 to 30 galls, 4=31 to 100 galls, and 5=more than 100 galls ( Taylor and Sasser, 1978 Taylor and Sasser, 1978 ). 2.10 Data analysis 2 Statistical data analysis was carried out with the open-source software R Version 2.14.1. The significance of differences among treatments was determined by Duncan's multiple range test (DMRT). According to DMRT at P ≤0.05, means depicted by the same letter are not significantly different. Dose-response probabilistic (Probit) models were used on J2s mortality data to determine the lethal concentration of MSC filtrates that kill J2s of M. javanica by 50% (LC 50 ). 3 Results 1 3.1 SEM of the mustard seed cake extract 2 SEM images provided important foundational observations to acquire morphological alterations in the target samples. The SEM images were studied closely to observe the surface morphology. Fig.2 Fig.2 shows the SEM images of the SE and NSE at different magnification levels. Figs. 2 Figs. 2 a‒2d show pictures of an NSE sample (control set-up). These images represent smooth surfaces without pores or fissures and intact surface structures, firmly bonded to each other. In comparison, pictures of an SE depict uneven and disrupted microstructures ( Figs. 2 Figs. 2 e‒2h). 3.2 Ultrasonication impacts on the production of secondary metabolites and identified mass profiles 2 By taking advantage of these ultrasonic treatments, we identified 17 secondary metabolites, including nematostatic compounds, in the extract of MSC. The LC-ESI-MS spectra of SE are presented in Figs. 3 3 and 4 4 . These compounds were identified as 3-methylsulfinyl propyl isothiocyanate, sinapine, 1-methoxybrassitin, 4-hydroxybenzyl glucosinolates, patuletin 3-gentiobioside, isorhamnetin 3,7-di- O -‍β‍-glucopyranoside, rutalexin, benzeneacetaldehyde, 5-methylthiopentylglucosinolate, campesterol, coumestrol, methyl oleate, 4-hydroxyglucobrassicin, gluconapin, Se-methylselenocysteine, methoxybrassinin, and indole-3-acetonitrile, and their corresponding peak ion masses were found at m / z 163.1, 310.2, 496.2 [M+2H] +‍ , 458.2 [M+CH 3 OH+H] +‍ , 656.2, 682.3 [M+ACN+H] +‍ , 274.3 [M+ACN+H]‍ + , 162.2 [M+ACN+H]‍ + , 477.1 [M+ACN+H]‍ +‍ , 400.1, 301.2 [M+CH 3 OH+H]‍ +‍ , 338.3 [M+ACN+H] +‍ , 885.5 [M+ACN+2H]‍ +‍ , 372.1 [M‍-‍H] -‍ , 545.1 [3M-H]‍ - , 265.2 [M-H]‍ -‍ , and 311.1 [2M-H] - , respectively (Figs. 3 3 and 4 4 ). Among identified compounds, sinapine was found as the most abundant compound, followed by gluconapin, with a relative percentage of peak area of 34.5% and 32.2%, respectively (Tables 1 1 and 2 2 ). The percent peak areas of all identified compounds of the SE sample compared with those of the NSE sample are illustrated in Tables 1 and 2. For example, the peak areas of identified compounds in SE were 13.0% (isorhamnetin 3,7-di- O -‍β‍-glucopyranoside), 8.4% (5-methylthiopentylglucosinolate), 8.2% (patuletin 3-gentiobioside), 7.8% (rutalexin), and 6.2% (3-methylsulfinyl propyl isothiocyanate) at retention time of 8.03, 14.72, 7.86, 11.44, and 1.14 min, respectively, in positive mode of LC-ESI-MS. In negative mode, we also noted the percent peak areas as 32.2% (gluconapin), 16.5% (methoxybrassinin), 8.2% (Se-methylselenocysteine), and 2% (indole-3-acetonitrile), respectively, at retention time of 1.47, 14.12, 6.17, and 19.92 min. At the same retention time and instrumental set-up, the percent areas of peak ions were observed to be lower in the NSE than in the SE. Thus, it is worth considering that the identified compounds in SE show a higher percentage of peak area. 3.3 In vitro nematostatic effects of sonicated extract and non-sonicated extract samples on J2s mortality 2 Table 3 Table 3 shows the variation in the rate of J2s death in the aqueous concentration of extract at different incubation periods. We observed that J2s mortality was nil in water (control). In the parallel study, the mortality rate of J2s was highest (65.00%) at 18-h incubation period in a 35-ppm concentration of SE, while the least mortality (6.25%) was seen in 5-ppm NSE. The pattern of J2s death found was in direct proportion to the concentration strength and incubation period in both types of extract samples. All the concentrations of both SE and NSE showed an impact on J2s mortality; however, the mortality rate of J2s was much higher in different concentrations of SE. Values expressed as mean±standard error of four measurements, with different superscript letters within groups, are significantly different ( P ≤0.05). MSC: mustard seed cake; SE: sonicated extract; NSE: non-sonicated extract; J2s: the second stage juveniles; ppm: parts per million; DW: distilled water. 2 Treatment 2 Incubation period (h) 5 Mortality of J2s nematodes (%) 5 ppm 15 ppm 25 ppm 35 ppm DW 3 SE 6 13.75±1.49 d 22.00±1.29 c 33.50±1.70 b 45.50±1.93 a 0 12 21.75±1.37 d 32.25±1.49 c 42.25±1.49 b 56.75±2.01 a 0 18 31.50±1.04 d 42.00±1.47 c 51.75±1.75 b 65.00±1.58 a 0 3 NSE 6 6.25±0.85 d 13.75±1.10 c 22.25±1.49 b 30.75±1.25 a 0 12 9.50±1.04 d 18.00±1.29 c 27.00±1.68 b 37.50±1.70 a 0 18 12.25±1.10 d 23.25±1.49 c 33.50±1.70 b 43.00±1.47 a 0 3.4 LC 50 of sonicated extract and non-sonicated extract samples against J2s of M. javanica 2 The LC 50 value was determined for mortality in J2s after immersion in SE and NSE samples at three different durations of exposure: 6, 12, and 18 h. The LC 50 values of SE at 6, 12, and 18 h of exposure duration were 51.76, 29.79, and 13.34 ppm, respectively, for J2s mortality, while for NSE, the respective LC 50 values were 116.95, 76.38, and 55.59 ppm at a similar exposure time. This result indicates that the value of LC 50 was higher to kill half of the J2s population during the short exposure time, while for a longer duration of exposure, the obtained value of LC 50 was lower ( Table 4 Table 4 ). The LC 50 response of SE was observed to be most effective in J2s mortality compared to the tested LC 50 response of NSE at a similar duration of exposures ( Table 4 Table 4 ). Probit analysis was performed to obtain LC 50 with 95% confidence limits. LC 50 : lethal concentration of MSC filtrates that kill J2s of M. javanica by 50%; MSC: mustard seed cake; J2s: the second stage juveniles; SE: sonicated extract; NSE: non-sonicated extract; ppm: parts per million; Probit: probabilistic. 2 Treatment 3 LC 50 with 95% confidence limit (ppm) 6 h 12 h 18 h SE 51.76 29.79 13.34 NSE 116.95 76.38 55.59 3.5 In vitro reversibility test of J2s 2 This test was conducted to understand the recovery activity of paralyzed J2s of M. javanica in the SE sample. The J2s incubated in SE were found incapable of movement on the first day after transfer to the water. Still, mobility in J2s significantly increased when J2s were maintained in water for a week. The data of percent paralyzed J2s are shown in Fig. 5 Fig. 5 at the 1st, 3rd, 5th, and 7th days. However, we observed that mobility in J2s did not return to levels comparable to the control at the end of the week. The results indicated that the SE could contain a high enough capacity of nematostatic compounds to kill the J2s. In this study, we also noted that if the incubation period of treatment is considered short, then treated J2s, after washing in water, would recover from paralysis within a week or two. 3.6 Root-knot index assessment in tomato roots 2 In a pot experiment, J2s-infected roots of tomato plants were analyzed. Specifically, the RKI was considered as the basis for analysis. We observed that the roots were severely knotted in the experiment's control set-up, while fewer and smaller root knots were developed in the root dip-treated tomato plants with SE and NSE. The RKIs of the control plants, NSE-treated plants (35-ppm), and SE-treated plants (35-ppm) were 4.65, 1.50, and 0.94, respectively ( Table 5 Table 5 ). We also noted that the RKI in plants treated with SE was significantly reduced with increased concentrations. Various levels of NSE also limited the RKI, although less effectively. Interestingly, an SE of 35-ppm concentration used as a root-dip treatment significantly ( P ≤0.05) controlled root-knot infection in tomato roots ( Table 5 Table 5 ). The RKI data of tomato plants are shown as mean±standard error ( n =4). Means with the same letter are not significantly different ( P > 0.05) at the probability level, according to Duncan's multiple range test. RKI: root-knot index; SE: sonicated extract; NSE: non-sonicated extract; ppm: parts per million. Treatment Concentration (ppm) RKI 4 SE 5 3.070±0.047 c 15 2.710±0.031 b 25 1.850±0.064 e 35 0.940±0.027 g 4 NSE 5 3.850±0.028 b 15 3.070±0.047 c 25 2.650±0.064 d 35 1.500±0.129 f Control 4.650±0.064 a 4 Discussion 1 Natural compounds are generally enclosed in rigid and insoluble structures such as vacuoles, bilayers, lignin, and cell walls ( Corrales et al., 2009 Corrales et al . , 2009 ). These factors have prevented the extraction of secondary metabolite compounds from seed cakes by straightforward extraction methods. This is why we used the ultrasonication technique to enhance the extraction efficiency of secondary nematostatic compounds. Seventeen secondary metabolites, including nematostatic compounds, were identified in the SE sample. The ultrasonic features assisted in releasing the metabolites in SE. Similar observations have also been reported in other seed cake samples, such as defatted hemp, flax, and canola seed cakes ( Teh and Birch, 2014 Teh and Birch, 2014 ). The effectiveness of ultrasound power was observed in enhancing polyphenol extraction. Willis (2017) Willis (2017) reported approximately 400 000 extant species of vascular plants on earth, which are the source of hundreds of thousands of metabolites whose utility has not been well explored. Ultrasound technology will undoubtedly augment their role as a sustainable approach, having the additional capacity to extract an enormous repertoire of specialized (or secondary metabolite) compounds ( Alseekh and Fernie, 2018 Alseekh and Fernie, 2018 ). In vitro results demonstrated that when the concentrations of extract of 5, 15, 25, and 35 ppm were tested individually against J2s of M. javanica , a precise compelling level of extract strength was observed. These results are the first to demonstrate the contact nematostatic potential of the SE of MSC. The mortality of J2s was severe when exposed to SE because of the high extraction efficiency of compounds in the samples via ultrasonic treatment. The results are in accordance with earlier reports ( Corrales et al., 2009 Corrales et al . , 2009 ; Ntalli and Caboni, 2012 Ntalli and Caboni, 2012 ; Dahlin and Hallmann, 2020 Dahlin and Hallmann, 2020 ), which reported that mustard plants and their by-products possess antinematode and antimicrobial properties. In the literature, plant by-products belonging to the family Brassicaceae are probably the most cited due to the presence of isothiocyanate and related hydrolysis products, which include a broad spectrum of biocidal activities ( Matthiessen and Kirkegaard, 2006 Matthiessen and Kirkegaard, 2006 ; Agerbirk and Olsen, 2012 Agerbirk and Olsen, 2012 ; Ntalli and Caboni, 2012 Ntalli and Caboni, 2012 ; Dahlin and Hallmann, 2020 Dahlin and Hallmann, 2020 ). Sinapine, a choline ester of sinapic acid, is one of the significant phenolic choline esters and occurs at high concentrations in Brassicaceae seeds ( Zhou et al., 2005 Zhou et al., 2005 ), which was also detected in this study as a significant compound. The antinematode properties of this phenolic compound have also been reported by Vaganan et al. (2014) Vaganan et al . (2014) . The most considerable concentration was found to be 35 ppm with strong killing properties (LC 50 =51.76, 29.79, and 13.34 ppm at 6, 12, and 18 h exposure, respectively ( Table 4 Table 4 )). Thus, the collective activity of SE makes MSC likely to be a more promising plant-based natural agent acting against nematodes. Pot experiment results demonstrate that the impacts of all SE treatments were significant compared to the NSE when applied as a bare-root dip treatment in tomato roots that were exhibited ( Table 5 Table 5 ). The mustard plants contain glucosinolate compounds and their derivatives, including isothiocyanates. These compounds have a marked anesthetic effect on nematodes ( Ntalli and Caboni, 2012 Ntalli and Caboni, 2012 ; Dahlin and Hallmann, 2020 Dahlin and Hallmann, 2020 ). Our results are strongly in agreement with previous reports of the active suppression of nematodes in soil amended with plant by-products of the family Brassicaceae ( Matthiessen and Kirkegaard, 2006 Matthiessen and Kirkegaard, 2006 ). A single mustard plant can contain several different types of glucosinolates. The types and quantities of GSLs are highly variable among the species and even among the cultivars of mustard ( Kruger et al., 2013 Kruger et al . , 2013 ). These nematostatic compounds are generally safe in our ecosystem ( Ntalli and Caboni, 2012 Ntalli and Caboni, 2012 ). 5 Conclusions 1 We determined the strength of the MSC extract solution, which can be strongly stimulated by ultrasound features and has shown potent antagonistic activity to J2s of M. javanica . The nematostatic action was due to the high extraction efficiency of nematostatic compounds in the SE sample. A significant alteration was also observed in SEM images on the surface of ultrasonic-assisted MSC, which might be due to the phenomenon of the microstreaming effect. Sonication treatment induces a microstreaming effect and can enhance mass transfer produced on cavitational bubble collapse. This, in turn, results in the cell wall destruction of plant tissues, thus providing better contact and interactions of solvents in and out of the plant materials. Indeed, ultrasonic features supported the extraction efficiency in the target sample. 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App Energy , 114 : 909 - 923 . https://doi.org/10.1016/j.apenergy.2013.08.085 https://doi.org/10.1016/j.apenergy.2013.08.085 Alternative methods of microorganism disruption for agricultural applications Zhou H , Huang YX , Hoshi T , et al. , 2005 . Electrochemistry of sinapine and its detection in medicinal plants . Anal Bioanal Chem , 382 ( 4 ): 1196 - 1201 . https://doi.org/10.1007/s00216-005-3241-1 https://doi.org/10.1007/s00216-005-3241-1 Electrochemistry of sinapine and its detection in medicinal plants Hera NADEEM and Faheem AHMAD conceived and designed the experiments, interpreted the results, and drafted the manuscript. Hera NADEEM performed the experiments and analyzed the data. Pieter MALAN, Amir KHAN, Mohd ASIF, Mansoor AHMAD SIDDIQUI, and Simon TUHAFENI ANGOMBE commented and critically revised the manuscript. All authors have read and approved the final manuscript. All authors have full access to data in the study and take responsibility for the integrity and security of the data. This work was supported by the University Grants Commission (UGC-BSR Research Start-up-Grant: F30-409/2018), India. 10.1631/jzus.B2000746 1673-1581（2021）07-0563-12 2020-11-24 2021-01-10 2021-07-15 2022-05-09 2021-05-12 © Zhejiang University Press 2021 Hera NADEEM, Pieter MALAN, Amir KHAN, Mohd ASIF, Mansoor AHMAD SIDDIQUI, Simon TUHAFENI ANGOMBE, and Faheem AHMAD declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 7 22