Euphorbia trigona latex nematicidal activity on the root-knot nematode Meloidogyne

The aim of this study was to test nematicidal activity of the Euphorbia trigona latex on Meloidogyne incognita juveniles and to partially purify and characterize three proteases present in this latex. Three distinct proteases were partially purificated from E. trigona latex. They were named here trigonin 1, 2 and 3. Their molecular weights were estimated at: 36, 31 and 29 kDa, for trigonin 1, 2 and 3, respectively. The pH and temperature that provided highest protease activity were pH values of 4.0, 6.0 and 9.0, and 70 °C. The crude extract containing the three proteases present in E. trigona latex reduced significantly (p < 0.01) the number of live M. incognita juveniles in 96% after 24 hours treatment. The present study is the first report of E. trigona latex with nematicidal activity, therefore more studies regarding latex proteases action on nematodes are needed.


Introduction
Euphorbia is a cosmopolitan genus which shares in common the characteristics of having specialized, highly reduced, flowerlike inflorescences, and the presence of milky white latex (Fonseca et al., 2010;Gunawardana et al., 2015).Euphorbia trigona is a succulent plant from Africa, known as African milk tree because of its high latex production, cultivated in various countries for ornamental purposes (Villanueva et al., 2015).Many studies suggest that the latex of plants from Euphorbia genus is a rich source of proteases (Yadav et al., 2012;Badgujar & Mahajan, 2013;Mahajan & Adsul, 2015;Rezanejad et al., 2015;Flemmig et al., 2017).Recently, our research group reported for the first time proteases from E. milii latex with nematicidal activity on Panagrellus sp.larvae (Sufiate et al., 2017).However, there are no studies of the E. trigona latex regarding nematicidal action.
Meloidogyne spp.(root knot nematodes) are responsible for reducing the production of several plants with economic importance, causing many losses to agriculture.Although chemical nematicides are efficient in nematodes control, they are extremely toxic and non-specific (Adegbite, 2011).Thus, there is the need to develop new eco-friendly strategies to combat these nematodes.
Therefore, the aim of this study was to test nematicidal activity of the E. trigona latex on Meloidogyne incognita juveniles and to partially purify and characterize three proteases present in this latex.

Latex obtainment
Euphorbia trigona latex was collected by means of superficial cuts on plants from Viçosa, Minas Gerais, Brazil.The latex was collected in microtubes and immediately stored at -20 °C.After five hours, the latex was thawed at room temperature, and the clear supernatant was collected, which was denominated as crude extract.

Obtaining of Meloidogyne incognita juveniles
Pure population of Meloidogyne incognita collected in Lavras, Minas Gerais, identified by analysis of esterase phenotypes (Carneiro and Almeida, 2001), were maintained in soybean plant during 60 days.After this period, the root system of the plants was submitted to Baermann funnel for hatching eggs and obtaining second stage juveniles (J2), which were quantified in Peters' chamber.The nematode suspensions were calibrated to 50 J2/mL.

Protease and protein assay
Protease activity was measured (Soares et al., 2013).One protease unit was defined as the amount of enzyme required to release 1.0 g of tyrosine per minute under the assay conditions.Protein content was determined according to Bradford (1976).

Purification
The crude extract was applied in a gel filtration column Sephacryl® S-300 previously equilibrated with citratephosphate buffer 25 mM (pH 6.0), at 4 °C.The flow was adjusted to 0.5 mL/min.Protease elution was monitored by protease activity and by protein content.Fractions with high protease activity were pooled, constituting the proteases partially purified (PPP).SDS-PAGE 10% was used to monitor the purification (Laemmli, 1970), and the gel was stained with coomassie blue.
A zymogram was prepared from a PAGE (10%) containing casein 0.1% co-polymerized.The gel was incubated in citrate phosphate buffer 100 mM (pH 6.0) at 70 °C during 30 minutes.Then, it was stained with coomassie blue, and the protease activity was observed due to halos.

PPP characterization
PPP activity was determined in different pH values, ranging from 2.2 to 10.0, using citrate-phosphate buffer 100 mM (pH 2.2 to 8.0), and glycine-sodium hydroxide buffer 100 mM (pH 8.0 to 10.0).For temperature effect characterization in the protease activity, different temperature values ranging from 40 to 80 °C were utilized.
PPP activity was measured in presence of the following inhibitors at 10 mM concentration: iodoacetamide, phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetraacetic acid (EDTA).This assay was conducted using citratephosphate buffer 100 mM (pH 3.0 and 6.0), and glycine-sodium hydroxide buffer 100 mM (pH 9.0), at 70 °C.All the protease activity assays were performed in triplicate.

Nematicidal assay
The effect of E. trigona latex on M. incognita juveniles was tested.Two groups were formed in microtubes, a treated group containing crude extract and approximately 50 M. incognita J2, and a control containing the same number of M. incognita J2, without latex crude extract.This assay had seven replicates for each group.The microtubes were incubated at 28 °C, in a dark room, during 24 hours.After this period, the number of live M. incognita J2 was counted in each tube of both groups.For data analysis, analysis of variance was used at significance levels of 1 and 5%.The destruction efficiency of M. incognita juveniles in relation to control was evaluated by Tukey test at 1% significance level.Subsequently, the percent reduction of juveniles' number was calculated according to the following equation: Reduction= ( juveniles from controljuveniles from treatment) x 100 % juveniles from control

Results and Discussion
The purification methodology allowed partially purification (Table 1) of three distinct proteases, which were named here trigonin 1, 2 and 3 (Figure 1a).In the SDS-PAGE, there are three protein bands present in the pool formed after the gel filtration chromatography (Figure 1a).In the zymogram, there are also three degradation halos (Figure 1b), indicating that the three proteins present in the pool are three proteases.However, the methodology used in this study did not allow that each of the enzymes were purified separately.The molecular weight of proteases was estimated at approximately: 36, 31 and 29 kDa for trigonin 1, 2 and 3, respectively (Figure 1a).These molecular weights are similar to those related for hirtin from E. hirta (34 kDa) (Patel et al., 2012), eumiliin from E. milii (30 kDa) (Fonseca et al., 2010), and neriifolin from E. neriifolia (35 kDa) (Yadav et al. 2012) (1) One protease unit (U) was defined as the amount of enzyme required to release 1.0 g of tyrosine per minute under the assay conditions.Source: Authors.Source: Authors.
PPP showed highest activity at 70 °C.After this temperature, there was an abrupt decline in protease activity (Figure 2a).In relation to the pH effect on protease activity, the pH values of 4.0, 6.0 and 9.0 resulted in highest activity, also suggesting the presence of three enzymes (Figure 2b).Source: Authors.
PMSF inhibited PPP activity in 35, 84 and 77% at pH values 3, 6 and 9, respectively.Iodoacetamide reduced PPP activity in 34 and 31% at pH 6 and 9, respectively, and it had no effect on PPP activity at pH 3. EDTA presence was responsible for increased activity in 52% at pH 3 (Figure 2c).These results indicate presence of serine and cysteine proteases in E. trigona latex.Serine proteases were described in E. hirta and E. neriifolia (Patel et al., 2012;Yadav et al., 2012), and cysteine proteases were observed in E. microsciadia and E.nivulia (Badgujar & Mahajan, 2012;Rezanejad et al., 2015).
The crude extract containing the three proteases present in E. trigona latex reduced significantly (p < 0.01) the number of live M. incognita J2.The efficiency of E. trigona crude extract in nematodes reduction is showed by the high reduction percentage observed, with 96% reduction in 24 hours of treatment, when compared to control (Figure 3).In a similar study, the proteases from Synadenium grantii reduced the number of M. incognita juveniles in 100% after 24 hours of treatment (Gomes et al., 2019).The present study is the first report of E. trigona latex with nematicidal activity, therefore more studies regarding latex proteases action on nematodes are needed.

Conclusion
E. trigona latex contains three distinct proteases, which were named here trigonin 1, 2 and 3, with an estimated molecular weight of 36, 31 and 29 kDa, respectively.The results indicate that, among these three proteases, there are serine and cysteine proteases.The pH and temperature that provide highest protease activity are pH values of 4.0, 6.0 and 9.0, and temperature of 70 °C.The crude extract containing the three proteases from E. trigona latex has nematicidal activity on M. incognita J2 larvae.

Figure 1 .
Figure 1.a) Purification analysis of proteases from Euphorbia trigona latex by SDS-PAGE 10%.Lane M: Protein molecular weight markers; Lane 1: crude extract; Lane 2: proteases partially purified.Dashed arrows indicate the three proteases from E. trigona latex.b) Zymogram of the proteases partially purified from E. trigona.Dashed arrows indicate the clear bands formed by proteases activity.

Figure 2 .
Figure 2. Characterization of the proteases partially purified from Euphorbia trigona.a) Temperature effect on the proteases partially purified activity from Euphorbia trigona.b) pH effect on the proteases partially purified activity from E. trigona latex.c) Effect of inhibitors on enzymatic activity of proteases partially purified from E. trigona latex.

Figure 3 .
Figure 3. Average number of Meloidogyne incognita J2 after 24 hours treatment with the extract obtained from Euphorbia trigona latex.The control had no enzymes.Asterisk indicates significant difference (p < 0.01) between the treated group and the control by Tukey test at 1% significance level.