Different sources of silicon in the embryonic development and in the hatching of Meloidogyne javanica

Silicon is the main objective of many researchers whose goal is to see its use as an inductor resistance. The action of the inductor happens in the plants, where it does not affect the pathogen directly, but the effect will contribute for its control. Therefore, the main objective of this report was to evaluate the effect of silicon on the embryonic development and the hatching of Meloidogyne javanica. For this, three sources of silicon (Silifort ® , Rocksil ® and wollastonite) in the dosage 0 (distilled water), 1⁄2, 1 and 2x the producer indication, resulting in a factorial 3 x 4, were evaluated in two different times. In Petri dishes were add 1 ml of suspension with 1500 eggs, and eventually juveniles of M. javanica, and 9 ml of treatment, which were evaluated, in Peters chambers, the percentage of eggs one, two, tetra and multicellular, eggs containing juvenile formed, and the hatched in a period of three days and seven days after the incubation (DAI). The wollastonite had stimulated the embryonic developments, as the increase of the dosage, were not seen with the Silifort ® and Rocksil ® experiments. The increase in the dosage of wollastonite caused stimulus to the hatching in 3 DAI, while Silifort ® caused the reduction of hatching in 3 DAI in both experiments. In 7 DAI, there was a reduction of hatching of juveniles exposed to Silifort ® and Rocksil ® , in the experiments 1 and 2, respectively.


INTRODUCTION
Silicon is one the most abundant nutrients in Earth, losing only for oxygen, being found in silica ways and silicate (Sripanyakorn et al., 2005).In the soil solution, it is found especially in the non-dissociated monossilicic acid, which is absorbed by the plants (Takahashi, 1995).Some studies present the element as an alternative to disease control in different pathosystems (Mendonça et al., 2013;Ramos et al., 2013;Santos et al., 2014), including the root-knot nematode (Dutra, 2004;Guimarães et al., 2008;Oliveira et al., 2012), in which *Corresponding author.E-mail: dani.mattei@hotmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License there are indications that the nutrient acts as a resistance inducer (Guimarães et al., 2010;Silva et al., 2010).However, to be considered as an inducer, the product cannot show direct action over the pathogens, but it should be limited to the expression of genes and to the biochemical mechanisms of the plant (Métraux, 2001).Besides, some inducing agents have direct and/or indirect action on the nematode, as the plants extract and the rhizobacteria (Remamoothy et al., 2001;Franzener et al., 2007), can act in different stage of the parasite cycle, including the hatching.
The hatching of nematode is a complex process, which is influenced for many ambient factors or which is linked to the nematode (Dias-Arieira et al., 2008).Some substances, natural or artificial, can detain or advance the hatching process, influencing the soil inhabitants' survival (Kraus and Vander Louw, 1996;Byrne et al., 2001;Charlson and Tilka, 2003).Chemicals used in agriculture can also have a direct effect in the process of hatching.For example, the change of soil pH and the liberation of ammonium ions affected the hatching and the permeation of Meloidogyne incognita (Kofoid and White), Chitwood and Heterodera glycines Ichinohe (Sudirman and Webster, 1995;Zasada and Tenuda, 2004).Silva (2009) observed that the incubation of M. incognita eggs for 14 days in a solution containing 5 and 10 µg/ml of Adicarbe reduced hatching of second-stage juveniles, which, according to the author, can be attributed to interruption or delay in embriogenic development of the parasite.The permeability of the eggshell, which regulates the entrances of water and the hydration of second-stage juveniles (J2), also can influence in the hatching (Jones et al., 1998).
Despite the reports that show the silicon as a resistance inductor, it is not known if it has some action on embrionic development and hatching of nematode.Therefore, it was aimed to evaluate the direct action in different sources and doses of silicon in the embryonal and in the hatching of juveniles of M. javanica (Treub) Chitwood.
Eggs from M. javanica were extracted from pure population, kept in tomatoes roots, in greenhouse, according to the Hussey and Barker (1973) methodology, adapted by Boneti and Ferraz (1981), and suspension was counted in Peters' chamber, on optical microscope, and calibrated for 1500 eggs/ml.The evaluation and  the count of eggs in different periods of embryonal and hatching of juveniles were done.The products solution were prepared by homogenization in distilled water, using 0 (control), ½, 1 and 2 x the doses that was recommended by the producer as: 5 ml/L (0.50%) and 20 g/L (2.00%) for Silifort ® and Rocksil ® , respectively.For wollastonite, it was used the recommended doses of 0.7 g/L (0.07%), considering that the relative power of neutralization (PRNT) consists in 79.26% and the sufficient quantity for the correction is 1.4 ton/ha, in a surface of 20 cm.
The hatching chambers consisted in Petri dishes (100 mm of diameter x 15 mm of height), which received 1 ml of nematodes suspension and 9 ml of silicon suspension.After this, they were closed and placed in plastic trays, being added to the bottom of the trays a slide with 0.5 cm of water, to keep the humidity of the Petri dishes, avoiding the evaporation of the solution.They were kept under temperature of 26.0° ± 2.0°C, in which the agitation of the plates was done once a day, to promote oxygenation.The evaluation was done three days and seven days after the incubation (DAI), being analyzed 1 ml aliquot of each plates' solution, in Peters chambers, underneath optical microscope, classifying the eggs as one, two, tetra and multicellular, with juvenile formed inside of egg or hatched juvenile.
The data obtained from the evaluation were used to calculate the difference between the percentage of eggs in each period of development and the hatching of juveniles and this difference was put in modulus.After this, the data were transformed by √(x+1) and they passed through variance analysis at 5% level of probability.When there was interaction among the factors, the doses were studied inside the products for regression analyzis.When there was significance only for the isolated ones, the products were compared by the test of Tukey at 5% level of probability and the doses for regression analysis is being adopted as the linear and quadratic, in function of a better fit (R²).

RESULTS AND DISCUSSION
The analysis of variation showed that in the experiment 1, the factor product was significant for eggs bi and multicellular in 7 DAI and for eggs with juveniles and juvenile hatching in both periods of evaluation, while the doses was significant only for eggs with juveniles in 7 DAI.The interaction between the factors product x doses was significant for eggs unicellular and eggs with juveniles at 3 DAI and for juvenile hatching in 3 and 7 DAI, and, in these situations, the studies of doses into the products continue.In experiment 2, the factor product was significant for eggs unicellular, eggs with juveniles and hatched juvenile in both periods of evaluation, multicellular eggs and tetra-eggs in 7 DAI.The factor dose was significant for unicellular eggs in both evaluation and for bi and tetracellular in 7 DAI.The factor dose was significant for eggs unicellular in evaluations, eggs multistage and hatched juveniles in 3 DAI and eggs bicellular and eggs with juveniles in 7 DAI.The interaction between the factors product x dose was significant for eggs containing juveniles and hatched juveniles at 3 DAI, while in 7 DAI, there was a significant situation for uni, bi and eggs containing juveniles.
In the treatment only the product was significant; in general, Silifort ® reduced the embryonary development and the hatching, especially if it is compared to the wollastonite (Table 1).On the other hand, the wollastonite was the product that most stimulate the embryonary development and the hatching of juveniles.The Rocksil ® , however, presented different datas, being either similar to Silifort ® either to wollastonite (Table 1).The effect of different products about the embryonary development can be attributed to the chemical composition of them, as in variety as in percentage of elements.In the evaluated products, the wollastonite had a higher quantity of SiO 2 , with 52%, followed by Silifort ® and Rocksil ® (21.39 and 17.43%, respectively).However, the solubility of the source is also variable, being the Rocksil ® less soluble than potassium silicate (Teixeira et al., 2008).The Si solubility in the products is also variable, the wollastonite shows 4.6% of soluble Si (Ramos et al., 2009), while the Silifort ® showed 10% of soluble Si, according to the producer information.
Evaluating the results for each product, it is possible to show two hypothesis: the first presents that the product with a higher quantity of SiO 2 (wollastonite) stimulated the nematoide's embryonary development; and the second that the products with a higher quantity of Si soluble, inhibit the embryonary development and the hatching, probably because of the faster liberation of the solution.
When the doses was significant, but there was no interaction between the factors, it was possible to observed the increase of the number of the eggs with juveniles was directly proportional to the increase of the dose (in 7 DAI in experiment 1) with an adjust of the linear equation y=0.9013x+2.4134(R 2 =0.8659).A similar result was seen for unicellular eggs in 3 DAI in experiment 2 (y=0.3497x+3.714;R 2 =0.729).The number of multicellular eggs observed in 3 DAI in experiment 2 is related to the quadric equation 0.7227x 2 +1.9554x+1.8826(R 2 =0.8487), with the highest point in 1.35 times the recommended dose (these data are not presented).In this situation, the adjust to the ideal dose to get the best result is essential, once the increase in the activity of nematodes can induct the faster consumption of resources and it can damage the capacity of penetration in the radicular system (Steele and Hodges, 1975), besides this, it can disfavor the survive of the nematodes population when the application in absent of host plant is done (Viglierchio, 1991).
The increase of the dose, regardless of the product, resulted in a bigger difference in unicellular eggs and eggs with juveniles before and after the exposition to the treatment, which indicates a stimulus to the development.The salinization of the solution is one of the factors that can explain the results, once all the substance that is release in the solution at least one cation different from H + and one anion different from OH -it is considered as salt (Oliveira and Marmo, 2014), thus the silicon of calcium and/or magnesium can be considered as salt, because when they are in contact with water in soil they release Ca 2+ and/or Mg 2+ and SiO 3 2- (Nolla, 2004).The exposition of nematode to the solution with ions with potential to increase the salinity changed the orientation of juveniles (Le Saux and Queneherve, 2002) and reduced the M. javanica development (Prot, 1998).This data is opposite to the ones that were observed in this study and it can be related to the population variability, once the permeability of the eggs is dependent on the constitution of two or three lipid layers under the chitin layer that constitutes the shell (Jones et al., 1998).
In the analyze of the development of the factors, it was possible to see that in the Experiment 1, in 3 DAI, the wollastonite reduced the percentage of unicellulars eggs when added until the dose of 0.83 from the one recommended by the producer.On the other hand, eggs containing juveniles only presented a reduction when used maximum dose.The percentage of hatched juveniles decreased to the dose of 0.75 from the recommended by the producer, with a late increase (Table 2).In Experiment 2 (Table 3), in 3 DAI, the application of wollastonite promoted an increase of hatching to the dose next of the recommended, that suggests that higher doses stimulated the hatching.However, in 7 DAI, independent of the experiment and the dose, the wollastonite did not affect the hatching.This happened because of the fact that the M. javanica shows a higher tolerance to the salt solutions when compared to other species of nematode (Ismail and Hassabo, 2014).
The Silifort ® , in 3 DAI and in 7 DAI in Experiment 1, showed a linear reduction of hatched juveniles (Table 2).Besides, in Experiment 2, in 3 DAI, it reduced the number of eggs with juveniles and the hatching, especially when it was used in higher doses (Table 3).However, the Rocksil ® showed a linear reduction of the percentage of eggs with juveniles and hatched juveniles with the increase of dose (Experiment 2) (Table 3).These data corroborate the ones presented by Al-Sayed et al. (1986), who observed that the exposition of Meloidogyne to the salt solutions can result in a decrease of hatching.
By data analyzes variation of answer to the hatching of juveniles were submitted in different doses and products.Probably, the different ions, the concentration of solutions and the duration of exposure that the eggs were subjected to influenced in the results.The effect of ions in the process of hatching was suggested in an experiment that added ethylenediamine-tretracetic acid to the soybean root, and increased the juveniles hatching of H. glycines, suggesting that the compost removed the ions that influenced it in a negative way (Teff and Bone, 1985).
According to Al-Sayed et al. (2014), who studied the effect of water in the salt irrigation on the M. incognita population in different hosts, most concentrations of salts caused adverse effects in the plants growing and can increase the damages caused by nematodes in vivo, which could influence in the development and reproduction of nematode, being the damages variable, depending on nematode species, the concentration of salts and the host plant.Lehman et al. (1971)   al. (1958) had seen the effect of these ions to the increase that happens in the osmotic pressure, as a consequent reduction in hatching.However, the potassium nitrate (KNO 3 ) that also causes the increase of osmotic pression, cannot influence significantly the juveniles hatching of M. incognita (Surdiman and Webster, 1995).It is also important to remember that there could be a difference between the promoted hatching in products test in vitro and in vivo, especially because of the soil solution complexity, as described for zinc, that disfavored the hatching of H. glycines in vitro (Teff and Bone, 1984;Behm et al., 1995), but did not show effect in vivo (Behm et al., 1995), probably because zinc is a cation that is adsorbed to the particles of soil (Clarke and Shepherd, 1966).Therefore, the realization of the tests in vivo to the confirmation of the data in this report is necessary before the decision of silicon indication to the direct management of M. javanica.

Conclusions
The wollastonite has stimulated with increase embryonary development of M. javanica, as the increase of the dose, being observed the opposite in Silifort ® and Rocksil ® .The wollastonite has also stimulated the juveniles hatching of M. javanica, especially when it was used in higher dosages.Silifort ® and Rocksil ® have reduced the embryonary development in a higher application of the dosage, and so they can be used as an alternative to inhibit the hatching.

Table 1 .
Percentage of the numbers of eggs uni, bi, tetra and multicellular, containing juveniles and with hatched juveniles, in different treatments, in 3 and 7 days after the built of the hatching chamber when compared to the initial percentage of inoculate, in Experiment 1 and Experiment 2.
bRates followed by the same letter in columns are not different according to the Tukey test at 5% level of probability.Original data transformed by √(x+1) for statistic analyze.

Table 2 .
Development of interaction between treatment and dose considering the percentage of eggs number in different stage of development or hatched juveniles in three and seven days after the incubation (3 and 7 DAI) when compared to the initial percent age in inoculate (Experiment 1).