Plant–parasitic nematodes are recognized as the causes of serious yield losses on a wide range of crops [1]. The most destructive species is Meloidogyne incognita which cause serious problem in various agricultural crops. Root–knot nematode, Meloidogyne incognita Kofoid and White Chitwood (Tylenchida: Heteroderidae) is a major plant parasitic nematodes affecting quantity and quality of the crop production in many annual and perennial crops. Infected plants show typical symptoms including root galling stunting and nutrient deficiency, particularly nitrogen deficiency [2]. M. incognita causing an estimated yearly crop loss of $100 billion worldwide [3]. Nematodes are difficult to control because of their wide host range and high rate of reproduction, with females capable of producing up to thousandeggs/ female [4]. Chemical control is expensive and is economically viable only for high value crops and creates a potential hazard to the environment and human health [5]. Biopesticides currently are integrated into many diverse agricultural production schemes. These materials can be effective and safe, but their use requires more sophistication than chemical pesticides on the part of the user. Many of these products have specific requirements for storage and application, and to treat them like a chemical pesticide often results in failure. As biological organisms they require appropriate biotic as well as abiotic conditions for success. Users would benefit from learning how to maximize efficacy before or after the organisms have been applied. Among microorganisms regulating nematode densities in soil, fungi hold an important position due to their parasitic, antagonistic or predatory behaviors. Some species have potentials in biocontrol and exhibit a range of antagonistic activities, including production of nematotoxic compounds [2,6,7]. Nematophagous fungi directly parasitize nematodes or secrete nematicidal metabolites affecting viability of one or more stages. The search for nematotoxic or antagonistic compounds in culture filtrates has greatly intensified in recent years, due to the number of toxins, enzymes or compounds derivable from their metabolites [8-10]. Inhibition of ChE activity is one of the best characterized biomarkers and has been intensively used in environmental studies, showing a specific response to organophosphate (OP) and carbamate pesticides [11,12]. Abamectin is a macrocyclic lactone derived from the soil bacterium Streptomyces avermitilis that has been shown to have nematicidal properties [13]. And a different mode of action than the other currently available nematicides [14]. It is suitable compound for seed treatment since it can be stored for several months while maintaining its nematicide properties. It can be applied to seeds at high concentrations, does not bio-accumulate and is not taken up by plants [15]. Cadusafos is an organophosphorus nematicide under the trade name ‘‘Rugby, Cadusafos controls a wide range of plant parasitic nematodes, such as Tylenchulus semipenetrans [16]. ChE stands out as an ideal biomarker to evaluate agriculture-related pollution effects in the area. Further, it would be useful to trace a link between pesticide use, detection of residues in the environment and their toxic effects AChE has been widely studied in many different species because it can be inhibited by Ops and CAs. ATP ase is a group of enzymes that play an important role in intracellular functions and that are considered to be a sensitive indicator of toxicity [17]. It can be target for many groups of pesticides. The aim of this is to investigates the effect of Organophosphates (cadusafos , fenitrothion) and the Biopesticides (abamectin, biofly) against root knot nematode Meloidogyne incognita also The potency of these pesticides to inhibit acetylcholine esterase ( AChE) and adenosine triphosphatase (ATPase ) activity of such root knot nematode.

Nematode cultures

The root-knot nematode M. incognita was isolated from infected roots of eggplant (Solanum melongena L.) obtained from El-Nubaria region, Behera Governorate, Egypt. Eggs and second-stage juveniles (J2) were extracted from infected roots by the sodium hypochlorite method [18].

Synthetic Insecticides

Abamectin: Group name: Avermectin (Biopesticide) Common name: Abamectin Trade name: Vabcomic Empirical formula: C₄₈H₇₂O₁₄ (80% avermectin B1a); C₄₇H₇₀O₁₄ (20% avermectin B1b) Molecular weight: 873.1 (avermectin B1a); 860.1 (avermectin B1b) Formulation: 1.8% E.C. Source: Hebei Veyong Bio-Chemical Co, ltd, china).

Biofly: Group name: Biological insecticide Common name: Beauveria bassiana Used and applied rate: Entomopathogenic fungus used for control of a wide range of coleopteran, homopteran and heteropteran pests. Fungus applied at rate of 100cm³/100liter water Trade name: Biofly Source: E1-Nasr Bio insecticides and Fertilizers Company, E1-Sadaat, Egypt.

Cadusafos: Group name: Organophosphorus, Common name. cadusafos Trade name: Rugby.Chemical name (IUPAC): S,S-di-secbutyl O-ethyl phosphorodithioate Empirical formula: C₁₀H₂₃O₂PS₂ Molecular weight: 270.4 Formulation: 20% E.C a Use: Agricultural Nematicide / Insecticide .Source: FMC Australasia Pty Ltd.

Fenitrothion: Group name: Organophosphorus Common name: Fenitrothion Trade name: Fentro Chemical name (IUPAC): O,Odimethyl O-4-nitro-m-tolyl phosphorothioate Empirical formula: C₉H₁₂NO₅PS Molecular weight: 277.2 Formulation: 50% E.C applied at rate of 250 cm³/100 liter water Use: insecticides Source: Agrochem, Alwatneia Company, Alex., Egypt.

Nematicidal assay on eggs

M. incognita was cultured in the greenhouse on tomato plants (New L-402, nematode susceptible) inoculated with a single nematode egg mass [18]. After 55 days, egg masses were hand-picked from galls of tomato roots and surface sterilized in 0.5% sodium hypochlorite for 3 min and washed with sterile water 3 Times. J2 were hatched from the egg masses, collected daily and Stored at 4°C. 2ml of egg masses of nearly uniform size were transferred to a 6cm-diameter autoclaved Petri dishes containing 2ml filtrate of different dilutions of (1.8% abamectins, 100% biofly 20% cadusafos and 50% fentrothion)the series of concentration were around recommended dose of each pesticides ,eggs maintained at the same volume of disteled water served as controls; three replicates of each treatment and control were included. Plate lids were sealed with parafilm and the plates were kept at 25°C. After 7 days hatched J2 were counted with the use of an inverted microscope and egg hatched reduction for a treatment was calculated as: average % of non-hatched J2 in the filtrate/average % non-hatch in control with sterilized water × 100.

Nematicidal assay second stage juveniles (J2)

2ml of the above solutions as previously described from each concentration was added to 2ml of nematode suspensions M. incognita second stage juveniles & in 50 ml glass capsule. A control treatment was made by adding 2ml of distilled water plus 2ml of the nematode suspension. Each treatment was replicated three times. The number oviable and dead nematodes was counted with the aid of a light microscope after 24, 48 and 72hrs at 25 ± 1°C and the nematode mortality was calculated for each treatment. Only the nematodes which did not regain motility were considered “dead”. The mortality percentage was calculated according to the Abbott’s [19] formula:

Whereas: m and n percentages mortality in treated sample and control.

Enzyme preparation

1ml, of nematode suspension or egg filtrate of each pesticides mixture stoked for 48 hrs from bioassay test were homogenized with ice cold buffer for 30 s and a small amount of glass beads (<106 l m, Sigma) were adding during homogenization with cooling to obtain at least 95% breakage. Homogenate was centrifuged. The clear supernatant was collected and kept frozen at -20°C until assayed.

Enzyme determination

Acetylcholinesterse (AChE) activity: The AChE was determined by the colorimetric method of Ellman et al. [20]. The suspension was homogenized in 0.1M phosphate buffer (pH 7.0). The homogenates were then centrifuged at 5,000 rpm for 20 min at 0°C. The supernatants were used as enzyme source for assay of AChE activity. Enzyme (150 uL), 100ul DTNB (0.01 M), and 30 uL ATChI (0.075 M) were added to 2.8 mL 0.1 M phosphate buffer (pH 8.0). The mixture was incubated at 37°C for 15 min. The absorbance was measured at 412 nm using Unico 1200 spectrophotometer. All of the treatments were done in triplicate. The specific activity of AChE was expressed as nmoles of acetylthiocholine iodide hydrolyzed/mg protein/min. Inhibition percentages of the activities against control were estimated in the enzymatic assay.

Total protein assay: Total protein was determined according to Lowry et al. [21]. This method was used to determine the protein content in nematode extract Protein extract (100 uL) was added to 2 mL alkaline copper reagent [48 of 2% (w/v) sodium carbonate in 0.1N sodium hydroxide 1 mL 1% (w/v) sodium-potassium tartrate 1 mL 0.5% (w/v) copper sulfate] and immediately mixed. After 10 min, 0.2 mL Folin-Ciocalteu phenol reagent was added and the samples were thoroughly mixed, then the absorbance of the developed blue color was measured at 600 nm using a Unico 1200 spectrophotometer. The protein content of the sample was determined by comparing to the standard curve of BSA.

Adenosine triphosphatase (ATPase) activity assay: ATPase activity was determined according to Koch [22]. After 48 h of bioassay test on the tested concentration, the nematode suspension was homogenized in Tris-HCl buffer (pH 7.4). The homogenates were centrifuged at 5,000 rpm for 10 min at -4°C. The supernatant was then centrifuged at 17,000 rpm for 30 min at -4°C. The pellets were resuspended in the same buffer. This suspension was used as enzyme source for the assay of ATPase activity. The enzyme activity was determined colorimetrically according to. Enzyme suspension was added to the reaction mixture that contained 100 mM NaCl, 20 mM KCl, 5 mM Mg₂Cl, and 5 mM ATP and the volume was adjusted to 850l L with Tris-HCl buffer (pH 7.4). This mixture was incubated at 37°C for 15 min and then stopped with 150 uL TCA. Four milliliters of fresh color reagent (5 g ferrous sulfate in 10 mL ammonium molybdate solution prepared in 10 N sulfuric acid) was added and absorbance was measured at 740 nm by using a Unico 1200 spectrophotometer. The enzyme activity was represented as micromoles inorganic phosphorus (Pi/mg protein/h). Inhibition percentages of the activities compared with control were considered in the enzymatic assay.

Statistical analysis

Data obtained were statistically analyzed according to SAS software program [23] Statistical analysis was performed using the SPSS 12.0 software program (Statistical Package for Social Sciences, USA). The log dose–response curves allowed determination of the LC50 values for the nematode bioassay according to probit analysis [24]. The 95% confidence limits for the range of LC50 were determined by leastsquare regression analysis of the relative growth rate (percentage of control) against the logarithm of the compound concentration. The data for AChE and ATPase activities were analyzed by one-way analysis of variance (ANOVA).

Toxicity of the tested pesticides to M .incognita

Results in (Tables 1a-1c and Table 2) indicated that all tested pesticides have toxic effect against M. incognita second-stage juveniles (J2 ) after 24 hrs of application while abamectin was the most toxic one with LC50 value 2.94 mg/L followed by 147.44 mg/L for fenitrothion, 976.77 mg/L for cadusafos while biofly was the least toxic one with LC50 value of 3190.18 mg/L. The toxicity increased after 48 hrs of treatment and the LC50 value were (1.68 & 34.69 & 163.13 and 877.98) respectively for abamectin, fenitrothion, cadusafos and biofly. The same trend of toxicity observed after 72 hrs of treatment abamectin was most toxic one followed by, fentirothion & cadusafos and biofly was least toxic with LC50 value (1.07, 20.64, 47.6 and 386.48) mg/L respectively. On the other hand the effect of the same tested solutions on Percentage of egg hatched reduction was calculated as: average % of non-hatched J2 in the filtrate/average % non-hatch in control with sterilized water × 100. Result in Table 2 indicated that abamectin was most effective in hatching reducing of M. incognita followed by cadusafos, fenitrothion and biofly was the least effective one with the LC50value (1.102. 308.04, 577.69, 620.62)mg/L, respectively.

Table 1a: Acute toxicity abamectin, biofly cadusafos and fenitrothion to M. incognita second stage juveniles (J2).

Table 1b: Acute toxicity abamectin, biofly cadusafos and fenitrothion to M. incognita second stage juveniles (J2).

Table 1c: Acute toxicity abamectin, biofly cadusafos and fenitrothion to M. incognita second stage juveniles (J2).

Table 2: A Acute toxicity abamectin, biofly cadusafos and fenitrothion to M. incognita egg hatching of nematode.

The in vivo inhibitory effect of abamectin, biofly, cadusafos and fentrothion to M. incognita Acetylcholinesterase (AChE) activity

The in vivo inhibitory effect of the above pesticides on AChE activity isolated from culture filtrate of M. incognita second stage juveniles (J2) and eggs filtrates was examined and the results are presented in Tables 3 and 4. Specific activity calculated as (n moles of acetylthiocholine iodide hydrolyzed/mg protein/min). Percentage of inhibition was calculated. It was observed that pesticides induced decrease in AChE activity compared with the control. Cadusafos was tested at concentration ranged between 300 to 6000 mg/L all concentration had inhibitory effect on AChE activity and the high inhibitory effect was found at 6000 mg/L it induced 94.81 inhibition the inhibition percentage are dose dependent (Table 3). Abamectin at concentrations ranged from 4.5 to 0,25 mg/ L decreases the activity of nematode second stage juveniles AChE in all tested concentration and the most inhibitory effect was 73.93 at 4.5 mg/L followed by biofly it decreased the activity of larval second stage enzyme to 68.28 at 2000 mg/L. It was observed that fenitrothion had least inhibitory effect on AChE activity and the percentage of inhibition was 57.19 at 1250 mg/L. data a (Table 4), illustrate the inhibition of AChE activity in M. incognita egg filtrates abamectin at 4.5 mg/L induced 73.93 while Cadusafos at 6000 mg/L induced 64.81 inhibition followed by biofly at 2000/mg L induced 62.73 inhibition fenitrothion induced only 44.34 inhibition at 1250 mg/L it was least effective on the in vivo effect inhibition of AChE activity in M. incognita egg filtrates than second stage juveniles (J2) It was observed that all the tested pesticides has an inhibitory effect on AChE activity and the inhibitory effect increased when the pesticides applied at the juveniles more than egg filtrates.

Table 3: The in vivo Inhibitory Effect of of abamectin, biofly, cadusafos and fenitrothion to M. incognita Acetylcholinesterase (AChE) Activity on second stage juveniles (J2).

Table (4): The in vivo Inhibitory Effect of of abamectin, biofly, cadusafos and fenitrothion to M. incognita Acetylcholinesterase (AChE) Activity on egg filterate after 7 days of treatment.

The in vivo effect of abamectin, biofly, cadusafos and fenitrothion to M. incognita ATPase activity.

Result indicated that abamectin was more effective than other pesticides on ATPase activity and the inhibition increase in case of egg filtrates than second stage juveniles (J2) it was 90.44 with egg filtrates at 4.5 mg/L of abamectin while it was 81.95 at the same concentration in case of juveniles (Tables 5 and 6). Biofly had the same inhibitory effect on ATPase activity and it was 81.98 at 2000 mg/L with egg filtrates and 78.80 at the same concentration with juveniles. Cadusafos and fentrothion had low inhibitory effect on ATPase activity in nematode egg filtrates and second stages juveniles comparing with abamectin and biofly.

Table 5: The in vivo effect of abamectin, biofly, cadusafos and fenitrothion to M. incognita ATPase activity second stage juveniles (J2).

Table 6: The in vivo effect of abamectin, biofly, cadusafos and fenitrothion to M. incognita ATPase activity egg filtrates.

This study illustrated the possibility of using abamectin and biofly ( biological pesticides) in controlling M. incognita instead of using the chemical pesticides to decrease the environmental pollutants of organophosphates, carbamates and other group of pesticides because the excessive use of organophosphates in agriculture has originated serious problems in the environment [25]. Although, these pesticides degrade quickly in water, there is always the possibility that residues and byproducts will remain, in relatively harmful levels in the organisms [26]. So this work indicates that abamectin the Bioproducts which based on pathogenic micro-organisms often referred as microbial pesticides; they are host specific and are potential candidates with regard to integrated pest management [27]. It has to be effective in controlling root knot nematode as cadusafos and more toxic than fenitrothion the organophosphate pesticides this result was in agreement with El-Nagdi and Youssef [28] who found that Abamectin significantly reduced the population density of M. incognita with increasing the measured plant growth. Also Nwadinobi et al. [29] reported that Abamectin is biocides reduced galling and delayed invasion and development of Meloidogyne spp. for 20 days when roots of 14 day old tomato seedlings were dipped in a low concentration of abamectin. The present study also investigates the toxicity of biofly the (biological pesticides) to nematode it act as the nematicides on its toxicity although it is less toxic than the other pesticides used but it had an inhibitory effect on both tested enzymes , it has an chemicals are inherently toxic. Therefore, alternative environment friendly measures are needed to be developed also biopesticides are often considered as one of the lowest impact on many beneficial organisms compared with other pesticides. They have attracted considerable attention recently for their inclusion in Integrated Pest Management (IPM) programs, but effects are highly variable depending on the species and studied developmental stage [30,31]. AChE activity is not due exclusively to organophosphates and carbamates, but those other classes of environmental contaminants such as complex mixtures of pollutants, detergents, and they also involved in AChE reduction [32-35]. There is general agreement that the toxic action of organophosphate and carbamate pesticides upon nematodes, insects and vertebrates is caused by their ability to inhibit acetylcholinesterase (AChE) in various parts of the nervous system, and thereby, disrupt nervoys transmission at that location [36]. So this illustrate why Cadusafos and fenitrothion had an inhibitory effect on AChE activity and why this enzyme used as biomarker for cadusafous and fenitrothion toxicity in nematode It was accepted that the mode of action of organophosphate (cadusafos) was reasonably certain that these compounds acted by the inhibition of acetylcholinesterase (AChE) at cholinergic synapses in the nematode nervous system. Inhibition of ACHE was most likely explanation for the observed effect of organosphosphate and carbamate nematicides on the orientation behavior of nematodes [37,38]. These chemicals perform their action by impairing nematode neuromuscular activity, thereby, reducing their movement, invasion, feeding and consequentially the rate of development and reproduction [39]. Bunt [40] suggested that these chemicals acted against the root-knot nematode by inhibiting egg hatching, their movement and host invasion by infective juveniles and checked further development of second stage juveniles that had penetrated the roots. Using AChE and ATP ase as biomarkers for the biofly and abamectin is considered to be a new trail to show whether this pesticides can follow this mode of action in nematode or not while results indicated that abamectin and biofly had an inhibitory effect on both enzymes this result was in agreement with Turner and Schaeffer [14] who indicated that although Abamectin is a macrocyclic lactone derived from the soil bacterium Streptomyces avermitilis that has been shown to have nematicidal properties and a different mode of action than the other currently available nematicides. Also Radwan [41] reported that Abamectin studied are needed in more precisely molecular level to strictly detect the mode of action of these pesticides. An explanation of this decrease of AchE activity could refer to the new mode of action of this biopesticides, which seem to work in a similar manner of other closely related compound (i.e., metabolites of actinomycetes) our result in agreement. This hyperactivity was different insect control agent which all of them caused either no change or a reduction in AchE activity. It seem as if it works in a reversible manner, producing an extra release of AchE which may prevents principally any message to be sent to the receptor and thus the insect become without neural orientation. Although the previously used Abamectin was believed to be of noncholinergic role, it seems that in the present study does. A hypothesis was offered to explain this decrease in AchE during the use of a closely related actinomycete derived compound Spinosad where Salgado [42] demonstrated that the receptors do so by mimicking the action of Ach at its binding site. Since Ach cannot then bind, such compounds don’t enhance and usually antagonize the response to currently applied Ach. Spinosad, instead of depressing the Ach response, greatly prolongs its duration. The ability of spinosad to prolong the action of AchE indicates that it and Ach can act simultaneously and therefore that studied are needed in more precisely molecular level to strictly detect the mode of action of biopesticides as abamectin and biofly which holds much promise to control insects in a novel mode of action. ATP ase is a group of enzymes that play an important role in intracellular functions and that are considered to be a sensitive indicator of toxicity [17]. Detection of ATPase inhibition could prove to be an important index for tolerable levels of a large group of environmental contaminants [43]. In the present study, the statistical tests performed on the data represent that biofly the biological pesticides and abamectin as biopesticides has modern mode of action on inhibiting such enzyme in nematode while the organophosphates pesticides cadusafos and fenitrothion had low inhibitory effect on ATP ase this related to their mode of action as an organophosphates and their action related to the inhibition acetylcholinesterase (ACHE) at cholinergic synapses in the nematode nervous system [37,38]. Inhibition of ACHE was most likely explanation for the observed effect of organosphosphate and carbamate nematicides on the orientation behavior of nematodes. Finally, it could be concluded that the results from this study indicated that using of both organophosphates and biopesticides achieved a highly activity against the root-knot nematode, M. incognita. Therefore, the results imply that it should focus on using biological agents as a safety method for human and environment to management the root-knot nematode in Egypt.