Evaluation of vEvaluation of Various Seed Extracts for Their Nematicidal Ecacies Against Root Nematode, Meloidogyne Incognitaarious Seed Extracts for Their Nematicidal Ecacies Against Root Nematode, Meloidogyne Incognita

Nematicidal activity of seed extracts of eight medicinally important plants viz. Abrus precatorius Linn., Amaranthus virdis Linn., Bunium persicum Boiss., Dioscorea deltoidea Wall. Ex Griseb., Teraxacum ocinale Weber., Malva neglecta Wall., Podophylum hexandrum Royle and Robina pseudoacacia Linn. against Meloidogyne incognita was evaluated in vitro at (24, 48 and 72 h) and greenhouse method. After 48 h of exposure, the in vitro results of second stage juveniles (J2s) displayed almost all extracts possess positive effects on J2 mortality. However, dominant mortality was observed by T. ocinale 93.67% and B. persicum 89.66% at 72 h exposure. In case of greenhouse effect extracts of T. ocinale and B. persicum reduced infestation as compared to inoculated control. The root knot index varied between 1-3 and egg mass index 0-3 as compared to control. This study suggested that seed extracts of these plants can be used for the management of M. incognita and could be used in replacement of synthetic nematicides.


Introduction
Agriculture is one of the important backbones of food production in the world. Due to rapid increase in world population and damage caused by various microorganisms and nematodes on various plants and vegetables which leads pressure on agriculture and ultimately production of various foods. Nematodes are very much responsible for the damage of plants and industrial, edible crops. The various species of Meloidogyne causes 90% loss in agronomical damages caused by root-knot nematodes [1]. Additionally these nematodes reacts with other disease causing organisms which leads to development of more complex diseases and loss of resistance to plants against pests and nematodes [2]. The development of new nematicides is very di cult task, because the living place of most nematodes is soil or plant roots.
The target of any chemical substance is very away from its application, more over cuticle and surrounding surface of nematode is impermeable to chemical substances, so it leads to toxicity, environmental pollution and an average farmer cannot afford them easily [3,4]. There is recent trend in search for the discovery of various antimicrobial, antioxidant and nematicidal agents from plant sources which are less expensive, eco-friendly, biodegradable, safer and natural [5][6][7][8][9]. Hence taken in consideration of the vast abundance of with tremendous traditional and biological use of Abrus precatorius L., Amaranthus virdis L., Bunium persicum Boiss., Dioscorea deltoidea Wall., Malva neglecta Wall., Podophylum hexandrum Royle, Teraxacum o cinale Weber., and Robina pseudoacacia L. from Kashmir [6] these plants were selected for present study.

Soxhlet extraction of seeds
The seeds of all worked plants were air dried and then ground into powder. The coarsely powder (40 g) of each plant in the form of packets was transferred into a 250 ml Soxhlet apparatus. About 180 ml of solvent chloroform and methanol (50:50, v/v) was taken and the apparatus was placed in 80 °C water bath and extracted for 3 hr. After complete extraction the solvent is removed with rota vapor and extract of each plant was collected. The yield of each extract (%) was obtained by following formula:

Nematicidal activity 2.3.1 In vitro method
The seed extracts of viz., P. hexandrum, B. persicum, A. precatorius, T. o cinale, A. virdis, D. deltoidea, M. neglecta and R. pseudoacacia were used for nematicidal activity to evaluate the effectiveness on M. incognita juveniles (J 2 ) mortality under different exposure time (24, 48 and 72 hours). In this experiment seed extracts were taken in sterilized petri dishes while tap water served as a control [10]. Hundred freshly second stage juveniles (J 2 ) of M. incognita, reared in the greenhouse in eggplant roots as pure culture, were added to each petri dishes by a pipette. Each treatment was triply replicated. All the dishes were kept randomly in laboratory at room temperature. Number of dead nematodes was observed at 24, 48 and 72 hours. Percentage mortality was calculated as: [mean of number of dead juveniles in treatment/total number of juveniles in treatment] × 100. After 72 h treatment the juveniles were transferred to distilled water for 24 h to ensure no recovery will occur.

Greenhouse method
This experiment is based in accordance to Holbrook et al. method [11]. Ten days old tomato seedlings were planted in clay pots with steam sterilized sandy loam soil. The plants were inoculated with counted number of infective juveniles (500 juveniles in 10 ml of water) and 100 ml of each plant extract separately by making holes of 3-5 cm deep near base of the plant. The holes were plugged with soil soon after inoculation. Watering of plants was done on regular basis. The plant inoculated with nematode without plant extracts serve as inoculated control. Each treatment had three replicate and experiment was repeated twice. After 30 days of inoculation, the plants were uprooted and washed gently with tap water. Number of galls per plants was counted visually. Number of egg masses per root system was counted by staining the infected root with phloxin-B (Holbrook). Disease intensity was determined by root-knot index and egg mass index on 0-5 rating scale according to [12].

Statistical analysis
Treatment results were statistically evaluated by using analysis of variance (ANOVA) by using the statistical software (IBM SPSS statistics software 20). Duncan's multiple range tests (DMRT) were performed to determine the differences between groups and means were considered signi cant at 5% signi cance level.

Yield of seed extracts
The Soxhlet extraction is one of the simple, useful and well known universal extraction technique. In seed extraction the polarity of solvent and time required for extraction process plays an important role in extraction e ciency. As displayed in (Table 1) the percentage yield obtained of all extracts were good however, highest yield was obtained in P. hexandrum 67.9% followed by R. pseudoacacia 57.5%, A. virdis 56.1%, M. neglecta 50.4 and T. o cinale 40.3%. The lowest yield was observed in seed extracts of D. deltoidea 23.9% followed by A. precatorius 13.8% and B. persicum 12.4%. In this work the Soxhlet extraction gives comparatively good extraction e ciency of seed extracts that can play an important role in relation to their nematicidal nature.

Nematicidal activity
All the seed extracts tested were observed to exhibit good level of inhibition towards juveniles of M. incognita in vitro method, but the level of toxicity varied with increase in exposure time. It has been found that all extracts showed higher mortality rates following 72 h. Data presented in the (Table 2) showed that all the seed extracts showed suppressive activity against M. incognita which increases in increasing exposure time. However, dominant inhibition was shown by T. o cinale (93.67%, 61.44% and 38.71%) and B. persicum (89.66%, 56.55% and 35.06%) mortality of M. incognita at 72, 48 and 24 hours respectively. The extracts of M. neglecta (75.28%, 55.73% and 36.67%) and R. pseudoacacia (70.39%, 48.66% and 36.43%) showed good inhibition of M. incognita while as normal effect was displayed by A. precatorius (67.62%, 41.19% and 34.33%), A. virdis (62.46%, 45.32% and 32.24%), D. deltoidea (62.37%, 42.22% and 31.47%) and P. hexandrum (60.31%, 40.33% and 34.53%) mortality of M. incognita at 72, 48 and 24 hours respectively. The results were con rmed by seed extracts of Ricinus communis and Peganum harmala which were used by Hasan et al. [13], Curto et al. [14] and Rich et al. [15] against Meloidogyne juveniles, and found them effective.
Laquale et al. [16] earlier reported that the leaf and root extracts of T. o cinale showed 36 and 50% juvenile mortality and 14.8 and 23.8% egg hatchability reduction of M. incognita. Khan et al. [17] found that various extracts of plants from apiaceae family such as Coccinia grandis, Commelina benghalensis, Leucas cephalotes, Phyllanthus amarusand, Trianthema portulacastrum displayed good nematicidal activity against M. incognita.
In case of greenhouse method as presented in (Table 3) T. o cinale and B. persicum extracts were found to be more potent than in reducing number of gall and number of egg masses in comparison to all other treatments. The seed extracts of M. neglecta, A. precatorius and R. pseudoacacia showed good inhibition of nematodes while as Lowest reduction was noticed in case of P. hexandrum and D. deltoidea as compared to control.

Conclusion
This study highlighted the nematicidal activity of eight seed extracts against root nematode, M. incognita. Both in vitro and green house method of nematicidal activity revealed that seed extracts especially those of T. o cinale and B. persicum extracts were very potent in suppressing nematode infestation. From this study it is concluded that the extracts of these plants can be used as a source of nematicidal agents in future drug, design and development.
Declarations Tables   Table 1. List of plant species used for chloroform and methanol (50/50, v/v) extraction, with their yields obtained by Soxhlet extraction.