THE POTENTIAL OF FIVE ECO-BIORATIONAL PRODUCTS ON THE REPRODUCTION OF ROOT-KNOT NEMATODE AND PLANT GROWTH

This work aimed to select potentially useful eco-biorational product that could be used to reduce the reproduction of root-knot nematode. The experiment was carried out in pots under net house. The results revealed that the bioproduct Dipel® (Bacillus thuringiensis ) proved to be the most effective treatment that reduced the root galls and egg masses by 71.60 and 77.78%, respectively. Also, Dipel® (B. thuringiensis) & Bio-nematon® (Paecilomyces lilacinus) showed their superiority between all treatments on the shoot, root length and root weight.


INTRODUCTION
Tomato plant (Solanum lycopersicum) is an important vegetable crop for nutritive sources such as carbohydrates, minerals and vitamins in Egypt (Howeedy et al., 2003). The most serious problems that threaten cultivated tomatoes are pests and diseases. The plant parasitic nematodes (PPN) have been found to be the most common and destructive diseases in the last two decades, and one of the most difficult plant diseases to control. The latest statistics showed that the estimated losses induced by PPN were $118 billion worldwide (Atkinson et al., 2012). There are thousands of nematodes genuses, but the most destructive genus around the world is the root-knot nematodes (Meloidogyne spp.). Meloidogyne spp can parasite on more than 2000 host species including vegetables, fruit trees, oil crops, fiber crops, grains crops and leguminous crops, next to weeds which is considered secondary host to nematodes (Khalil, 2013a). The most well-known species of root-knot nematode are Meloidogyne incognita, M. javanica, M. arenaria and M. hapla, which are responsible for high economic losses to varied crops. A number of methods for the root-knot nematodes management have been applied, and different levels of successes were displayed on crop protection (Randhawa et al., 2001 &Jain 2001). It was necessary to find alternatives and / or new approaches to manage and eliminate the plant nematodes diseases. The soil-inhabiting fungus Paecilomyces lilacinus (Thom) Samson (Eurotiales: Trichocomaceae) is capable of parasitizing nematode eggs, juveniles and females resulting in reduced soil population densities of plant parasitic nematodes (Atkins et al., 2005 andKhalil et al., 2012b). Furthermore, Trichoderma spp. plays major roles in controlling the plant diseases in roots, soil and foliar environments (Thangavelu and Mustaffa, 2012). Also, the bacterium Bacillus thuringiensis Berliner produces parasporal crystalline proteinaceous inclusions. Most of these crystal proteins or δendotoxins are toxic to larvae of lepidopteran, dipteran or coleopteran insects (Knowles and Dow, 1993), pathogenic protozoa, mites and nematodes (Fettelson et al., 1992). Meanwhile, it was reported that some strains of Bacillus subtilis have exhibited enormous potential as biocontrol agents in the management of root-knot nematodes (Karanja et al., 2007). Therefore, the objective of this study was to testify the efficiency of the commercial products as an alternative nematicides.
The nematode inoculation: The tomato plants were infected with root-knot nematode eggs which isolated from the infested roots of the eggplant (Solanum melongena L.) that obtained from Rashid region, Behera Governorate, Egypt. Sodium hypochlorite (NaOCl) was utilized for isolation of nematode eggs from root galls according to Hussey and Barker (1973). Moreover, the roots were stained for 15 minutes in an aqueous solution of Phloxine B stain to detect the presence of nematode egg masses (Holbrook et al., 1983). The Pots experiment: The pot experiment was carried out using tomato plants cv. super strain B, the Pots were 15 cm in diameter and 20 cm in depth and each pot filled with 1kg of autoclaved artificial mixture soil {1clay: 2 sand (v/v)}. The isolated eggs of root-knot nematode were applied at the rate of 5000 eggs / pot. Six treatments were applied, next to untreated check and each treatment was replicated five times, and each replicate contains one plantlet. Fifty days after planting, the seedlings were uprooted and root systems were assessed for galling (number of galls/root system), and egg masses/root system, in addition to the shoot length, shoot weight, root length and root weight. Application of eco-biorational products: The tested products were applied to soil as one-time drench according to the recommended dose as following: Bio Zeid ® applied at the rate of 40 kg / fed; Bio Arc ® utilized at 40 kg / fed; Stanes sting ® at the rate of 1L /100L water; Bio-Nematon ® at the rate of 1.2 kg / 100L water and Nemathorin ® at the rate of 12.5 kg / fed. While, the suggested dose of Dipel ® was 3 kg / fed. All treatments were applied two days after infection. The tomato plants were fertilized by (N: P: K 18:18:18 + TE). Statistical analysis: Data of the present study were analyzed using variance test (ANOVA). The experimental design was a complete randomized design. The least significant differences (LSD) at the 5% level of probability were determined using a computer program Costat software (1988).

RESULTS
The impact of certain eco-biorational products on galls and egg masses formation were recorded in Table (1) and Fig. (1). The obtained results revealed that all treatments reduce the galls without any significant differences. B. thuringiensis reduced the gall formation by 71.60%, followed by B.subtilis, P. lilacinus, B. megaterium, T. album and fosthiazate that recorded 60.94, 58.58, 57.98, 52.65 and 51.50 % reduction, respectively. On the other hand, B. thuringiensis proved to be the most effective treatment which minified the egg masses by 77.78%, followed by P. lilacinus, T. album, B. subtilis, fosthiazate and B. megaterium which recorded 65.18, 63.33, 62.96, 59.27 and 57.04% reduction, consecutively. According to obtained data it was found that all treatments increased the shoot system growth significantly as compared with untreated check as shown in table 2. There were no significant differences on shoot height among all treatments in comparison with untreated check.

DISCUSSION
According to this study, the efficiency of tested ecobiorational products which can reduce the reproduction of root-knot nematode were seen by the suppressing of the galls and egg masses formation and enhancement of plant growth. B. subtilis and B. thuringiensis are considered the most well-studied bacteria against plant parasitic nematodes (Crickmore et al., 1998;Dawar et al., 2008;Radnedge et al., 2003;Radwan, 2007;Siddiqui and Mahmood, 1999). Ashoub and Amara (2010) (Huang et al., 2009;Huang et al., 2005b;Ji et al., 2006;Kloepper and Ryu, 2006;Lahlali et al., 2013;Siddiqui and Mahmood 1999;St Leger, 1995). On the other hand, Mena et al. (1996) recorded that the B. thuringiensis var. kurstaki controlled M. incognita and Radopholus similis Cobb on Cucurbita pepo. While, Radwan (1999) observed that the shoot and root length and fresh weight of tomato plants were increased in the presence of B. thuringiensis var. kurstaki and Oxamyl. B. thuringiensis (Bt) produces one or more parasporal crystal inclusions (Cry or d-endotoxins). These toxins are known to be toxic to a wide range of insect species (Feitelson et al., 1992). Some Cry proteins are also toxic to nematodes (Feitelson et al., 1992). To date, five Cry proteins (Cry5B, Cry6A, Cry13, Cry14A, Cry21A) known to be toxic to larvae of a number of free-living or parasitic nematodes (Crickmore et al., 1998;Marroquin et al., 2000 andWei et al., 2003). Additionally, a number of studies have reported direct antagonistic effects of other bacteria to pathogenic nematodes belonging to the genera Heterodera and Meloidogyne, included B. amyloliquefaciens, B. cereus, B. licheniformis, B. megaterium and B. thuringiensis.
In surveys have been conducted worldwide to detect fungal parasites of Meloidogyne spp. was found that there are more than 30 genera and 80 species of fungi such as Arthrobotrys spp., Monacrosporium spp., Fusarium spp., Aspergillus spp., Penicillium spp., P. lilacinus and Verticillium chlamydosporium, (Chen et al., 1996a;Godoy et al., 1983;Li et al., 1994;Roccuzzo et al., 1993 andWang et al., 2001). However, in China, the predominant fungal species which collected from plant roots and infested soil was P. lilacinus that represented 49.3% of the isolates during a survey (Sun et al., 2006). The antagonistic fungus P. lilacinus proved its activity against root-knot nematodes on varied crops. Several reports clarified that using formulated P. lilacinus reduced the formation of galls and egg masses (Udo et al., 2013). Meanwhile, Khalil et al. (2012b) confirmed that liquid Bio-Nematon ® (P. lilacinus) and Dipel 2x ® (B.thuringiensis var. kurstaki), were the most effective treatments which suppressed the galls by 66.67 and 60.15 %, respectively, while decreased the egg masses by 75.97% and 74.97%, consecutively. Also, Kiewnick and Sikora (2006) recorded that the fungal biocontrol agent, P. lilacinus strain 251 (PL251) was potential to control the root-knot nematode Meloidogyne incognita on tomato. The pre-planting soil treatment reduced root galling by 66% and number of egg masses by 74%. P. lilacinus was effective against the root knot nematode and significantly reduced the galls number, egg masses and eggs per egg mass. Moreover, the enhancement of plant growth (Ganaie and Khan, 2010;Oclarit et al., 2009;Prakob et al., 2007 andSiddiqui et al., 2001). The action of P. lilacinus against plant parasitic nematodes was interpreted in multitude investigations. Khan et al. (2006b) and Khan et al. (2004) recorded the directed penetration of fungal hypha to the female cuticle of M. javanica by transmission electron microscopy. While, Park et al. (2004) reported that P. lilacinus could be produce leucino toxin and other nematicidal compounds. In the laboratory test this fungus infested eggs of M. incognita and destroys the embryos within 5 days because of simple penetration of the egg cuticle by individual hypha aided by mechanical and/or enzymatic activities, in addition to killing juveniles and females of M. incognita and Globodera pallida (Jatala, 1986). It was mentioned that P. lilacinus caused substantial egg deformation in M. incognita, these deformed eggs never matured or hatched (Jatala et al., 1985) . The serine protease produced by P. lilacinus might play a role in penetration of the fungus through the egg shell of the nematode (Bonants et al., 1995 andKhan et al., 2004). Also, it was reported that T.viride reduced galls formation and egg masses of Meloidogyne incognita, infecting Okra (Kumar et al., 2012). Le et al. (2009) investigated the potential of Fusarium and Trichoderma isolates against M. graminicola in rice. The results showed that Trichoderma isolates reduced galls formation up to 38%, while Fusarium isolates reduced the galls by 29-42%. Furthermore, Krishnaveni and Subramanian (2004) and Sharma (1999) indicated that T. harzianum, T. viride and P. fluorescens were effective in controlling the plant parasitic nematodes. Kavitha et al. (2007) found that P. fluorescens, B. subtilis and T. viride showed a significant increase in the plant growth parameters. However, the phytonematodes are affecting the Trichoderma spp. through the production of volatile and nonvolatile toxic metabolites, antibiotics, viridin, viridian, gliovirin, glisoprenins, heptelidic acid and others (Vey et al., 2001). Fosthiazate which belong to organophosphate group is inhibit the acetylcholine esterase (AChE) in various parts of the nervous system of nematodes and provides a highly performance as systemic nematicide. The results in this study are in agreement with those obtained by other researcher (Giannakou et al., 2005;Pathan et al., 2005;Russo, et al., 2003;Saad et al., 2011) who found that fosthiazate was effective against RKN. Besides, Radwan et al., (2012) confirmed that fosthiazate was the most effective treatment against the root galls formation in compared with four granular nematicides namely, cadusafos, carbofuran, ethoprop and oxamyl. Also, all treatments increased the plant growth indices. Whilst, Kesba (2011) found that nemathorin® 10% G (fosthiazate) was the superior treatment which reduced the galls and egg masses between all other treatments.

CONCLUSION:
It could be concluded that application of formulated ecobiorational products were effective against the