Thymus Citriodorus (Schreb) Botanical Products as Ecofriendly Nematicides with Bio-Fertilizing Properties

In recent years, interest has surged in the development of plant extracts into botanical nematicides as ecofriendly plant protection products. Aromatic plants are maybe the most studied category of botanicals used in this direction and the yielding essential oils are obtained on a commodity scale by hydro distillation. Nevertheless, can the bioactivity of aromatic plants always be attributed to the terpenes content? What would it mean for soil microcosms to bear the treatment of an essential oil to cure against Meloidogyne sp.? Are there other extraction procedures to prepare more ecofriendly botanical products starting from an aromatic material? Lemon thyme is studied herein for the first time for its nematicidal potential. We compare the efficacy of lemon thyme powder, macerate, water extract and essential oil to control Meloidogyne incognita (Chitwood) and Meloidogyne javanica (Chitwood), and we additionally study the secondary effects on soil microbes and free-living nematodes, as well as on tomato plant growth. According to our results lemon thyme powder enhances tomato plants’ growth in a dose-response manner and when it is incorporated in soil at 1 g kg−1, it exhibits nematicidal activity at a 95% level on M. incognita. The water extract yielding from the same dose is nematicidal only if it is left unfiltered; otherwise only a paralysis effect is demonstrated but inside the soil the biological cycle of the pest is not arrested. The essential oil is good both in performing paralysis and biological cycle arrest, but it detrimentally lowers abundances of bacterial and fungal feeding nematodes. On the contrary, lemon thyme powder and unfiltered water extract augments the bacterial biomass, while the latter also increases the bacterivorous nematodes. Overall, the bio fertilizing lemon thyme powder and its unfiltered water extract successfully control root knot nematodes and are beneficial to soil microbes and saprophytic nematodes.

The EC 50 values established for M. incognita after immersion of J2 in test solutions of NEMguard for 1 and 2 days were 0.048 and 0.040 (% v/v of formulated product); while for M javanica the respective EC 50 values were 0.063 and 0.1753 (% v/v of formulated product) (data not shown). This means that, to some extent, M. javanica regained activity after 2 days of J2 immersion in the test solutions. According to the paralysis bioassays, a dose-response relationship was evident for all three lemon thyme extracts and paralysis activity increased over time ( Table 2). The best effective extract was Plants 2020, 9,202 4 of 12 the EO, followed by WEF and finally H, for M. incognita and M. javanica. Similar sensitivity of the paralysis effect was shown for both nematode species, as EC 50 values were calculated at similar levels. In all cases, paralysis established after 2 days of exposure was not reversible since paralyzed J2s never regained motility. When different lemon thyme products were used in the pot bioassay to assess the efficacy of 1% (w/w) P and the respective component EO, H and WE (filtered or not), the best effective treatment was the P and WE, which exhibited efficacy at the level of NEMguard (garlic extract) (Figure 1), namely 95, 94 and 98%, respectively. The EO followed and the H was last. Interestingly, the WEF did not exhibit any activity when applied in the pot experiment ( Figure 1) and this might have to do with the stability of the extract inside the soil. Nematicidal activity (± st. error) of different lemon thyme botanical products (EO: essential oil; H: hydrosol; WEF: water extract filtered; WE: water extract; P: powder and NEMguard) against Meloidogyne incognita based on female counts per gr of root, 45 days after treatment. Different letters above columns correspond to statistically significant differences between treatments. The results of the one-way ANOVA, regarding the effect of treatments on soil bacterial and fungal biomass, as well as on soil free-living bacterivorous and fungivorous nematodes, are given in Figure 2A,B and Figure 3A,B, respectively. Bacterial biomass was found to be higher in the EO, WE, P and garlic extract treatments and differed significantly compared to the control, WEF and H. On the contrary, the fungal biomass did not differ among the treatments. The EO treatment presented significantly lower abundances of bacterial and fungal feeding nematodes compared to the control. On the contrary, the WE treatment significantly increased the numbers of the bacterivorous nematodes, but not the fungivorous. Nematicidal activity (± st. error) of different lemon thyme botanical products (EO: essential oil; H: hydrosol; WEF: water extract filtered; WE: water extract; P: powder and NEMguard) against Meloidogyne incognita based on female counts per gr of root, 45 days after treatment. Different letters above columns correspond to statistically significant differences between treatments.

Effects of Lemon Thyme Powder (P), Essential Oil (EO), Hydrosol (H), Water Extract (WE) and Water Extract Filtered (WEF) on Soil Bacterial and Fungal Biomass, along with the Soil Microbial Feeding Nematodes
The results of the one-way ANOVA, regarding the effect of treatments on soil bacterial and fungal biomass, as well as on soil free-living bacterivorous and fungivorous nematodes, are given in Figures 2A,B and 3A,B, respectively. Bacterial biomass was found to be higher in the EO, WE, P and garlic extract treatments and differed significantly compared to the control, WEF and H. On the contrary, the fungal biomass did not differ among the treatments. The EO treatment presented significantly lower abundances of bacterial and fungal feeding nematodes compared to the control. On the contrary, the WE treatment significantly increased the numbers of the bacterivorous nematodes, but not the fungivorous. Mean values (± st. error) of soil bacterial and fungal biomass of the experimental plots (C: water control; EO: essential oil; H: hydrosol; WEF: water extract filtered; WE: water extract; P: powder and garlic extract), 45 days after treatment. Different letters above columns correspond to statistically significant differences between treatments, as revealed by one-way ANOVA (Tukey p < 0.05).

Figure 3.
Mean values (± st. error) of soil bacterivorous and fungivorous free-living nematode abundances of the experimental plots (C: water control; EO: essential oil; H: hydrosol; WEF: water extract filtered; WE: water extract; P: powder and garlic extract), 45 days after treatment. Different letters above columns correspond to statistically significant differences between treatments, as revealed by one-way ANOVA (Tukey p < 0.05).

Soil Amendment with Lemon Thyme Powder (P) for Biofertilizing: A Dose-Response
A clear dose-response relationship was established considering fresh weights of both the aerial part and the root of tomatoes treated with increasing amounts of P as shown in Figure 4. No phytotoxicity was provoked on the tomato plants, even at the highest test concentration of 10% w/w. Mean values (± st. error) of soil bacterial and fungal biomass of the experimental plots (C: water control; EO: essential oil; H: hydrosol; WEF: water extract filtered; WE: water extract; P: powder and garlic extract), 45 days after treatment. Different letters above columns correspond to statistically significant differences between treatments, as revealed by one-way ANOVA (Tukey p < 0.05).

Figure 1.
Nematicidal activity (± st. error) of different lemon thyme botanical products (EO: essential oil; H: hydrosol; WEF: water extract filtered; WE: water extract; P: powder and NEMguard) against Meloidogyne incognita based on female counts per gr of root, 45 days after treatment. Different letters above columns correspond to statistically significant differences between treatments.

Effects of Lemon Thyme Powder (P), Essential Oil (EO), Hydrosol (H), Water Extract (WE) and Water Extract Filtered (WEF) on Soil Bacterial and Fungal Biomass, along with the Soil Microbial Feeding Nematodes
The results of the one-way ANOVA, regarding the effect of treatments on soil bacterial and fungal biomass, as well as on soil free-living bacterivorous and fungivorous nematodes, are given in Figures 2A,B and 3A,B, respectively. Bacterial biomass was found to be higher in the EO, WE, P and garlic extract treatments and differed significantly compared to the control, WEF and H. On the contrary, the fungal biomass did not differ among the treatments. The EO treatment presented significantly lower abundances of bacterial and fungal feeding nematodes compared to the control. On the contrary, the WE treatment significantly increased the numbers of the bacterivorous nematodes, but not the fungivorous. Mean values (± st. error) of soil bacterial and fungal biomass of the experimental plots (C: water control; EO: essential oil; H: hydrosol; WEF: water extract filtered; WE: water extract; P: powder and garlic extract), 45 days after treatment. Different letters above columns correspond to statistically significant differences between treatments, as revealed by one-way ANOVA (Tukey p < 0.05).

Figure 3.
Mean values (± st. error) of soil bacterivorous and fungivorous free-living nematode abundances of the experimental plots (C: water control; EO: essential oil; H: hydrosol; WEF: water extract filtered; WE: water extract; P: powder and garlic extract), 45 days after treatment. Different letters above columns correspond to statistically significant differences between treatments, as revealed by one-way ANOVA (Tukey p < 0.05).

Soil Amendment with Lemon Thyme Powder (P) for Biofertilizing: A Dose-Response
A clear dose-response relationship was established considering fresh weights of both the aerial part and the root of tomatoes treated with increasing amounts of P as shown in Figure 4. No phytotoxicity was provoked on the tomato plants, even at the highest test concentration of 10% w/w. Mean values (± st. error) of soil bacterivorous and fungivorous free-living nematode abundances of the experimental plots (C: water control; EO: essential oil; H: hydrosol; WEF: water extract filtered; WE: water extract; P: powder and garlic extract), 45 days after treatment. Different letters above columns correspond to statistically significant differences between treatments, as revealed by one-way ANOVA (Tukey p < 0.05).

Soil Amendment with Lemon Thyme Powder (P) for Biofertilizing: A Dose-Response
A clear dose-response relationship was established considering fresh weights of both the aerial part and the root of tomatoes treated with increasing amounts of P as shown in Figure 4. No phytotoxicity was provoked on the tomato plants, even at the highest test concentration of 10% w/w. Interestingly, the biofertilizing effect was even greater at the test concentration of 10% w/w for lemon thyme if compared with Melia azedarach ripe fruit powder tested at the same test concentration. It has to be noted that Melia azedarach (Linnaeus) is a plant species proven to have significant biofertilizing properties based on our previous studies; thus, we chose to use it here as a reference for biofertilization.
Interestingly, the biofertilizing effect was even greater at the test concentration of 10% w/w for lemon thyme if compared with Melia azedarach ripe fruit powder tested at the same test concentration. It has to be noted that Melia azedarach (Linnaeus) is a plant species proven to have significant biofertilizing properties based on our previous studies; thus, we chose to use it here as a reference for biofertilization. Figure 4. Weight increase of aerial parts and tomato roots after treatment with lemon thyme powder (P) at 0.1 to 10% against Melia azedarach powder 10% (MFP) and water control (C). Data are presented as means of five replicates with standard deviations. Means followed by the same letter are not significantly different according to Tukey's test (p ≤ 0.05). Upper case letters correspond to statistical differences on fresh aerial parts weight. Lower case letters correspond to statistical differences on fresh roots weight.

Discussion
Our study showed that lemon thyme has great nematicidal potential against two plant parasitic nematodes and that at the same time it benefits soil microorganisms, free living nematodes and plant growth. Interestingly, the best effective lemon thyme products according to paralysis experiments were, in descending order of efficacy, the EOs followed by WEF and finally H, for M. incognita and M. javanica (Table 2). According to the chemical composition analyses (Table 1), the EOs, and to a much lesser extend H, are rich in constituent terpenes and in particular geraniol. In addition, other researchers have published similar composition analyses of lemon thyme EOs [15]. Indeed, the nematicidal potential of EOs and H can, to some extent, be attributed to geraniol content, since, according to our previous studies, geraniol showed paralysis activity on J2, and the EC50 value against M. incognita was calculated at 237 and 158 μg mL −1 after 24 and 28 h of immersion in test solutions [28]. Interestingly, geraniol was synergistic when in binary mixtures with trans-anethole and carvacrol [30], and carvacrol is indeed a chemical component of both EO and H according to Table 1; thus, it can synergistically attribute to the efficacy with geraniol. However, the efficacy of WEF exhibited in the paralysis bioassays (Table 2) was not manifested in the pot bioassays (Figure 1). On the contrary, if filtering is skipped and plant residues are included in the amendnment, then the WE intervenes with the parasites' biological cycle (Figure 1). Interestingly, the unfiltered WE, together with P, were the most effective treatments at arresting the biological cycle inside the host roots according to female counts per gr of plant tissue. These two botanical products, representing a powder and a macerate, are the most chemically complex treatments used in the study and their nematicidal activity may be based not only on the supplied toxicity of plant secondary metabolites, e.g., terpenes, but also on their decomposition products, the changes in physical and chemical properties of the soil and the supply of biological parameters exhibiting activity [31][32][33][34]. On the other Figure 4. Weight increase of aerial parts and tomato roots after treatment with lemon thyme powder (P) at 0.1 to 10% against Melia azedarach powder 10% (MFP) and water control (C). Data are presented as means of five replicates with standard deviations. Means followed by the same letter are not significantly different according to Tukey's test (p ≤ 0.05). Upper case letters correspond to statistical differences on fresh aerial parts weight. Lower case letters correspond to statistical differences on fresh roots weight.

Discussion
Our study showed that lemon thyme has great nematicidal potential against two plant parasitic nematodes and that at the same time it benefits soil microorganisms, free living nematodes and plant growth. Interestingly, the best effective lemon thyme products according to paralysis experiments were, in descending order of efficacy, the EOs followed by WEF and finally H, for M. incognita and M. javanica (Table 2). According to the chemical composition analyses (Table 1), the EOs, and to a much lesser extend H, are rich in constituent terpenes and in particular geraniol. In addition, other researchers have published similar composition analyses of lemon thyme EOs [15]. Indeed, the nematicidal potential of EOs and H can, to some extent, be attributed to geraniol content, since, according to our previous studies, geraniol showed paralysis activity on J2, and the EC 50 value against M. incognita was calculated at 237 and 158 µg mL −1 after 24 and 28 h of immersion in test solutions [28]. Interestingly, geraniol was synergistic when in binary mixtures with trans-anethole and carvacrol [30], and carvacrol is indeed a chemical component of both EO and H according to Table 1; thus, it can synergistically attribute to the efficacy with geraniol. However, the efficacy of WEF exhibited in the paralysis bioassays ( Table 2) was not manifested in the pot bioassays (Figure 1). On the contrary, if filtering is skipped and plant residues are included in the amendnment, then the WE intervenes with the parasites' biological cycle (Figure 1). Interestingly, the unfiltered WE, together with P, were the most effective treatments at arresting the biological cycle inside the host roots according to female counts per gr of plant tissue. These two botanical products, representing a powder and a macerate, are the most chemically complex treatments used in the study and their nematicidal activity may be based not only on the supplied toxicity of plant secondary metabolites, e.g., terpenes, but also on their decomposition products, the changes in physical and chemical properties of the soil and the supply of biological parameters exhibiting activity [31][32][33][34]. On the other hand, the bacterial biomass was found to be higher in the EO, WE, and P treatments, but the EO treatment presented significantly lower abundances of bacterial and fungal feeding nematodes (Figure 2). On the contrary, the WE treatment significantly increased the numbers of the bacterivorous nematodes ( Figure 3). This shows that WE and P are the most effective treatments Plants 2020, 9, 202 7 of 12 against root knot nematodes and also the most beneficial according to soil parameters assessed herein. Lastly, the treatment of P furnished a clear dose-response biofertilizing effect (Figure 4), allowing applications even at higher concentration levels, without the fear of phytotoxicity, which may be useful in high nematode population levels. Interestingly, tomato growth was even greater at the test concentration of 10% w/w of lemon thyme, if one considers the Melia azedarach ripe fruit powder treatment tested at the same dose as a control according to our previous studies [35]. To the best of our knowledge, this is the first report on the comparative assessment of different botanical products, all originated from an aromatic species, against M. incognita and M. javanica. Additionally, we studied the ecotoxicity of these products and their effects on the tomato plants, along with their nematicidal activity. To date, many studies have shed light on the nematicidal activity of aromatic species (17)(18)(19)(20)(21)(22)(23)(24)(25)(26) but no ecotoxicity data have been given at the nematicidal dose rates. In addition, in many cases the bioassays are performed in vitro so there are no phytotoxicity data. This is first step in the use of lemon thyme against RKN. We are now in the process of testing our hit lemon thyme products in open field studies.

Plant Material, Nematode Populations and Reagents
Thymus citriodorus was a kind offer from a local farmer (Etherikon, Greek Herbs) cultivating biological aromatic plants. The aerial parts were collected at the flowering stage and were dried in the absence of light at room temperature. Subsequently, they were sealed in paper bags and kept at room temperature, in the dark, until use. The dried aerial plant parts were further processed into fine particle-powder (P), to be used thereafter for all extraction procedures and pot bioassays. Voucher specimens were taken to the Department of Ecology, School of Biology, Aristotle University of Thessaloniki, Greece, for species identification. Petroleum ether was of high-performance liquid chromatography grade. All chemical standards were obtained from Sigma-Aldrich (Milano, Italy).
M. incognita and M. javanica populations, originating from two single eggmasses of Greek origin were reared on tomato (Solanum lycopersicum Mill.) cv. Belladonna. Freshly hatched (24 h) second stage juveniles (J2) were extracted according to Hussey and Barker (1973) [36] from 60 day-old (d) infested roots, to be used for the bioassays.

Essential Oil (EO), Hydrosol (H) and Water Extract (WE and WEF) Production
The dried lemon thyme was subjected to water distillation using a Clevenger apparatus (Winzer) for 3 h at a ratio of 1/10 (w/v). That is, one hundred grams of aromatic plant was added to a 2000-mL glass flask with 1000 mL of distilled water. The EO obtained was dried over anhydrous Na 2 SO 4 and stored in dark glass vial with Teflon-sealed caps at −20 ± 0.5 • C until use. The yield of EO determined on average over three replicates was 5 mL 100 g −1 of dry weight (data not shown). The remaining hydrosol in the glass flask was filtered and used fresh for bioassays while in part it was submitted to chemical analysis after 1/1 (v/v) fractionation in petroleum ether.
The water extract was produced by mixing the dried lemon thyme with distilled water at a ratio of 1/10 (w/v) and sonicated for 15 min. Thereafter, filtration took place in part of the extract through a Whatman no. 40 filter paper (Whatman International Ltd., Maidstone, England), while another extract, containing plant tissues, was kept. All samples were stored at −20 ± 0.5 • C until use for bioassays. Before chemical analysis all water containing extracts were subjected to 1/1 (v/v) fractionation in petroleum ether.

Chemical Analysis
Chromatographic separation and identification of the EOs main components was performed on a Trace GC Ultra gas chromatograph (Thermo Finnigan, San Jose, CA, USA) coupled with a Trace ISQ MS detector, a split-splitless injector, a Thermo Scientific™ TriPlus RSH autosampler (Rodano-Milan, Plants 2020, 9, 202 8 of 12 Italy) and an Xcalibur MS platform. The EOs were diluted 1:100 (v/v) with hexane and one microliter of the diluted samples were injected with a split ratio of 50:1 on a 5% phenyl methylsiloxane fused silica capillary column (TR-5MS, 30 m length, 0.250 mm i.d., film thickness 0.25 µm). The injector and transfer line were at 220 • C and 220 • C respectively, the interface was at 250 • C, and the electron energy in the electron impact was 70 eV. Helium was the carrier gas at a constant flow rate of 1 mL/min. The GC oven temperature was programmed as follows: 70 • C (held for 5 min), then increased to 240 • C at a rate of 8 • C/min, and held at final temperature for 15 min. After 5 min of solvent delay, a mass range of m/z 50-600 was recorded. Mass spectrometry acquisition was carried out using the continuous electron impact ionization (EI) mode. The peak area integration and chromatogram visualization were performed using Xcalibur processing program. Peak identification and mass spectra tick evaluation was performed using the NIST11 database as well as by comparison of retention indices (RI) for alkanes C9-C24 with the ones reported by Adams 2007 [30] (Table 2). The nematicidal activity of EO, H and WEF, in terms of J2 motility block, was studied, and the EC 50 values were calculated. For each testing material a separate dose-response was established using 5 test concentrations ranging from 100 to 1000 µL L −1 . Stock solutions of EO were made in ethanol, and working solutions were brought to volume with distilled water containing the polysorbate surfactant 20 (Tween-20). All other test solutions were prepared in water. Final concentrations of ethanol and Tween-20 in testing wells did not exceed 1 and 0.3% v/v, respectively. Distilled water and a carrier control, that is ethanol and Tween-20 at concentrations equivalent to those in the treatment wells, served as a control. Around fifteen J2 were used per treatment well in Cellstar 96-well plates (Greiner bio-one). The plates were covered and maintained in the dark at 28 • C. Border wells were used to check the vapor drift. Juveniles were observed with the aid of an inverted microscope (Euromex, The Netherlands) at 40 after 24 and 48 h and were ranked into two different categories: motile or paralyzed. After the last assessment (48 h), the nematodes were transferred into plain water, after washing in tap water through a 20 µm pore screen to remove the excess treatment substance(s), and they were assessed again after 24 h for motility regain. Nematodes that never regained activity were considered dead and the subsequent paralysis as nematidical activity. Paralysis treatments were replicated six times, and each experiment was performed twice.

Effect of Lemon Thyme Powder (P), Essential Oil (EO), Hydrosol (H), Water Extract (WE) and Water Extract Filtered (WEF) on the Biological Cycle of M. incognita
A clay loam soil with 1.3% organic matter and pH 7.8 was collected from a non-cultivated field of the University Farm. It was dispensed through a 3-mm sieve and was partially air dried overnight. Soil moisture (oven drying at 110 • C for 24 h) and maximum water holding capacity (MWHC) were measured according to Pantelelis et al., 2006 [37]. Next, the soil was mixed with sand at a ratio of 2:1 and the mix was separated into 6 plastic bags representing experimental treatments. Nematode inoculation was made with 2500 J2 kg −1 soil and an equal distribution of juveniles in soil was assured by mixing and incubation according to Ntalli et al., 2010 [28]. Based on preliminary trials, 1 g of P per kg of soil was nematicidal against Meloidogyne incognita at efficacy levels of 95%. To be able to understand if the activity of P is based in the contained EO, H, WEF or WE, we tested P against these fractions used individually, in a M. javanica control pot bioassay. In particular, the test concentration of each material per gr of soil was as follows: P 1 g kg −1 soil, EO 50 µL kg −1 soil, H 10 µL kg −1 soil, WE 10 µL kg −1 soil and WEF 10 µL kg −1 soil. We used 50 µL of EO kg −1 soil because that is the expected content of the EO (yield 5%) in 1 g of P. Likewise, we used 10 mL of H, WE and WEF because that is the amount of respective extracts that corresponds to 1 g P according to the extraction ratio of 1/10 (w/v). As a commercial control, we used the nematicide NEMguard SC (garlic extract) at the recommended dose for nematode control under field conditions (4 L ha −1 corresponding to 2 µL of formulated Plants 2020, 9, 202 9 of 12 product per kg soil). Seven-week old tomato plants, cv. Belladonna, were transplanted into the treated soil and, at the end of the parasite cycle, inside the host roots, the efficacy assessments determined the number of M. incognita females per gram of root at 10× magnification control. The experiment was replicated once, and the treatments were always arranged in a completely randomised design with five replicates. On the 40th day, after the experiment described in Section 4.5, samples were taken to assess the Phospholipid Fatty Acid (PLFA) content of our soil samples as described in detail by Ntalli et al. (2018) [35]. The bacterial biomass was found as the sum of the 15:0, i-15:0, a-15:0, i-16:0, i-17:0, 16:1ω7c, 16:1ω9c, 16:1ω9t, cy17:0 and cy19:0 PLFA biomarkers, while the fungal biomass was equal to the 18:2ω9,12 one [38,39].
Soil free-living nematodes were extracted from 100 mL of each composite soil sample based on Cobb's sieving and decanting method, as modified by S'Jacob and van Bezooijen (1984) [40]. We estimated the nematode abundance under a stereoscope, and then fixed them in 4% formaldehyde. From each sample, we randomly selected and identified 150 nematodes to the genus level [41]. Nematode genera were assigned to trophic groups according to [42]. In our plots, we found six bacterivorous genera (Acrobeloides, Cervidellus, Rhabditis, Heterocephalobus, Chiloplacus, Panagrolaimus) and three fungivorous genera (Ditylenchus, Aphelenchus, Aphelenchoides).

Soil Amending with Lemon Thyme Powder (P) for Biofertilizing: A Dose-Response
Appropriate amounts of P were mixed with the soil and sieved twice so as to achieve the concentrations of 0.1%, 0.5%, 1% and 5% w/w. A treatment with 10% Melia azedarach ripe powdered plain fruits served as control [35] since, according to our previous studies, it exhibits biofertilizing properties if incorporated in soil before transplanting the tomato plants. We also used a control treatment of water. The moisture content of the soil never exceeded 24% of the MWHC. After 24 h, the soil was used for transplanting 7-week old tomato plants, cv. Belladonna, into 200-g plastic pots and was maintained at 27 • C and 60% RH with a 16-h photoperiod. Each pot received 20 mL of water every 3 days, and the plants were uprooted and gently washed 40 days post experiment establishment. The weight of the aerial part along with the root was measured. The experiment was replicated once, and the treatments were always arranged in a completely randomized design with five replicates.

Statistics
Natural death/immobility was eliminated according to the Schneider Orelli formula [43]: corrected % = {(mortality% in treatment − mortality % in control)/(100 − mortality % in control)} × 100, and experiments were analyzed (ANOVA) and combined over time. Since ANOVA indicated no significant treatment by time interaction, means were averaged over experiments. Corrected percentages of paralyzed J2 were subjected to nonlinear regression analysis using the log-logistic equation proposed by Seefeldt et al. [44]: Y = C + (D − C)/{1 + exp[b (log(x) − log(EC 50 ))]}, where C = the lower limit, D = the upper limit, b = the slope at the EC 50 , and EC 50 = the test concentration required for 50% death/paralysis after removal of the control (natural death/paralysis). In the regression equation, the test concentration was the independent variable (x) and the paralyzed J2 (percentage increase over water control) was the dependent variable (y). The mean value of the six replicates per test concentration and immersion period was used to calculate the EC 50 value.
Considering the pot bioassays, and since the ANOVAs indicated no significant treatment by time interaction (between runs of experiment), means were averaged over experiments. The data from the pot bioassays were expressed as a percentage decrease in the number of females per gram of root corrected according to the control (water), using the Abbott's formula: corrected % = 100 × (1−(females number in treated plot/females number in control plot)). It was fitted in the log-logistic model, as for paralysis data, to estimate the concentration that caused a 50% decrease in females per gram of root (EC 50 value).
We used one-way ANOVA to indicate the effect of different treatments on soil bacterial and fungal biomass as well as on free-living bacterivorous and fungivorous abundances. In all analyses, means were compared by Tukey's test at p < 0.05.

Conclusions
There is a great need for eco-friendly nematode control tools. Efficacy, even at the regulatory level, is at times even less important than phyto-and ecotoxicity concerns. Consequently, both should be highlighted before suggesting tools and doses. This is a first report on the nematicidal potential of different aromatic products from Thymus citriodorus against M. incognita and M. javanica along with their side effects on soil microorganisms and free-living nematodes encompassing soil microcosms as well as nematode host plants. Lemon thyme powder and unfiltered water extract were the best active soil amendments that did not harm the microbes and saprophytic nematodes. This first acted as tomato bio-fertilizer in a dose response manner. Thus, our findings underline lemon thyme potential in an integrated pest-management program.