Evaluation of various seed extracts for their nematicidal efficacies against root nematode, Meloidogyne incognita

The current study was assumed to identify the novel nematicidal activity of chloroform and methanol (50:50, v/v) seed extracts of eight medicinally important plants viz. Abrus precatorius Linn., Amaranthus virdis Linn., Bunium persicum Boiss., Dioscorea deltoidea Wall. Ex Griseb., Teraxacum officinale Weber., Malva neglecta Wall., Podophylum hexandrum Royle and Robina pseudoacacia Linn. using the root-knot nematode Meloidogyne incognita in in vitro and greenhouse experiments. At 72 h exposure, the extracts were found to be highly nematostatic, where nematodes were completely paralyzed at 12 and 48 h of exposure. However, dominant mortality was observed by T. officinale 93.67% and B. persicum 89.66% seed extracts at 72 h. In greenhouse method, extracts of T. officinale and B. persicum extracts were found to be most potent in reducing number of galls (1.76 and 2.37) and number of egg masses (0.06 and 2.18) respectively as compared to inoculated control. The root knot index of all extracts 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 upon further isolation and purification of bioactive substance responsible for nematicidal activity.


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 react with other disease causing organisms which lead to development of more complex diseases and loss of resistance to plants against pests and nematodes [2]. In last decades, the synthetic nematicides were the only available drugs used against these parasites, because of their indiscriminate use which leads to harmful side effects and consequently problems to the environment [3,4]. Furthermore, the development of resistance of microbes to these drugs is a major setback to their continued use in livestock and humans [5,6]. The development of new nematicides is very difficult task, because the living place of most nematodes is soil and plant roots. The target of any chemical substance is very away from its application. Moreover, cuticle and surrounding surface of nematode is impermeable to chemical substances, leads to toxicity, environmental pollution and because of their high cost an average farmer cannot afford them easily [7,8]. Hence, keep these things in consideration, the latest development is search for novel antimicrobial, antioxidant and nematicidal agents from plant sources which are less expensive, eco-friendly, biodegradable, safer and natural [9][10][11][12][13]. The report of WHO medicines obtained from plant sources serve about 80% of world population [14]. Hence, it is important to screen medicinally important plants which are used in ethanopharmacolgy or ethnomedicine. These plants may provide new, novel, inexpensive and above all locally available drugs especially for underdeveloped countries.

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 h. 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:

In vitro method
The seed extracts of viz., P. hexandrum, B. persicum, A. precatorius, T. officinale, A. virdis, D. deltoidea, M. neglecta and R. pseudoacacia were used for nematicidal activity to evaluate the effectiveness on M. incognita juvenile (J 2 ) mortality under different exposure time (24, 48 and 72 h). Briefly, 5 mL of nematode suspension containing 100 freshly second stage juveniles (J 2 ) of M. incognita, were put in vial with a final concentration of 100 ppm. These nematode vials were taken in eight petri dishes at 25 0 C. The seed extracts at a concentration of 1000 ppm of the test extract were prepared by dissolving different extracts in 0.5 mL dimethyl sulphoxide (DMSO), and to it add distill water to make final volume 5 mL. The extracts were then transferred in each eight sterilized petri dishes so as to cover whole surface. The Petri dishes were then kept for exposure at 24, 48 and 72 h. The volume of 0.5 mL DMSO with 9.5 mL distilled water served as control. 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 h. 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 [15]. 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 [16].

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 significant at 5% significance 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 efficiency. 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. officinale 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 efficiency of seed extracts which could play an important role in relation to their inexpensive cost nature in drug formulation.

Nematicidal activity
The worked plant extracts from Kashmir were used in vitro for the first time against plant parasitic nematode. 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. Data presented in the (Table 2) showed that all the seed extracts showed suppressive activity against M. incognita which increases in increasing exposure time. The highest mortality rate was observed at 72 h. The extract of T. officinale gave up to 93.67% immobilization activity when exposed for 72 h while (J 2 ) were innobilized up to 61.44% when exposed to 48 h in same extract. Laquale et al. [17] earlier reported that the leaf and root extracts of T. officinale showed 36 and 50% juvenile mortality and 14.8 and 23.8% egg hatchability reduction of M. incognita. The extract of B. persicum showed inhibition of 89.66%  [20] and Rich et al. [21] against Meloidogyne juveniles, and found them effective. Significant nematicidal activity of water extract of the Italian M. azedarach fruit pulp (IPWE) (EC50/48 h = 955 μg/mL) was obtained by Aoudia et al. [22] and among its active ingredient components were p-coumaric acid and p-hydroxybenzoic acid (EC50/48 h = 840 and 871 μg/mL, respectively).
In case of greenhouse method as presented in (Table 3), it has been observed that all seed extracts exhibit excellent nematicidal activity. Among the worked extracts, the T. officinale and B. persicum extracts were found to be more potent in reducing number of galls (1.76 and 2.37) and number of egg masses (0.06 and 2.18) respectively in comparison to all other seed extracts at (p < 0.05). The observed results are in complete agreement with the previous studies on roots and leaves [17]. The activity of other seed extracts showed good reduction of number of galls and egg masses as M. neglecta (5.04 and 4.06), A. virdis (4.25 and 8.08), A. precatorius (7.27 and 5.08) and R. pseudoacacia (8.09 and 6.17) respectively. However, the lowest reduction was noticed in case of P. hexandrum and D. deltoidea as compared to control (Table 3).

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 particularly those of T. officinale 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.