1 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.

Hence, to address the above mentioned issues chloroform and methanol (50:50, v/v) seed extracts of Abrus precatorius L., Amaranthus virdis L., Bunium persicum Boiss., Dioscorea deltoidea Wall., Malva neglecta Wall., Podophylum hexandrum Royle, Teraxacum officinale Weber., and Robina pseudoacacia L. from Kashmir were analyzed for nematicidal activity against Meloidogyne incognita.

2 Materials and methods

2.1 Source of seeds and chemicals

The seeds of worked plants were collected from nurseries in Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir (SKAUST-K) and various other parts of Kashmir in 2019. Dr. Sajad Gangoo authenticated the plants. The herbarium specimens were placed at Kashmir University Herbarium (KASH) under 823, 827, 891, 1374, 1357, 1391, 1398 and 1411 respectively for A. precatorius, A. virdis, B. persicum, D. deltoidea, M. neglecta, P. hexandrum, T. officinale and R. pseudoacacia of Voucher Specimen Numbers [Ref. No. F1 (Specimen voucher, CBT) KU/2019] and nematode Meloidogyne incognita were obtained from the section of plant pathology, Department of Botany, Aligarh Muslim University, Aligarh (India). The solvents and chemicals used were purchased from Sigma-Aldrich, USA.

2.2 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:

$$ {\text{Yield of an extract }}\left( \% \right) \, = \frac{{{\text{mass of extract obtained }}\left( {\text{g}} \right)}}{{{\text{mass of seed material }}\left( {\text{g}} \right)}} \times {1}00 $$

2.3 Nematicidal activity

2.3.1 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 (J2) mortality under different exposure time (24, 48 and 72 h). Briefly, 5 mL of nematode suspension containing 100 freshly second stage juveniles (J2) 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 0C. 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.

2.3.2 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].

2.4 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.

3 Results and discussions

3.1 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.

Table 1 List of plant species used for chloroform and methanol (50/50, v/v) extraction, with their yields obtained by Soxhlet extraction
Full size table

3.2 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 (J2) 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% juveniles at 72 h while the same extract showed 56.55% inhibition of M. incognita at 48 h exposure. Khan et al. [18] has performed the same study and they found that various plants from from apiaceae family such as Coccinia grandis, Commelina benghalensis, Leucas cephalotes, Phyllanthus amarusand, Trianthema portulacastrum displayed good nematicidal activity against M. incognita which validates the good nematicidal activity of B. persicum extracts. 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 h respectively. The results were confirmed by seed extracts of Ricinus communis and Peganum harmala used by Hasan et al. [19], Curto et al. [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).

Table 2 In vitro nematicidal activity of seed extracts of P. hexandrum, B. persicum, A. precatorius, T. officinale, A. virdis, D. deltoidea, M. neglecta and R. pseudoacacia against Mincognita.
Full size table

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).

Table 3 Green house method for determination of nematicidal activity of seeds extracts of P. hexandrum, B. persicum, A. precatorius, T. officinale, A. virdis, D. deltoidea, M. neglecta and R. pseudoacacia against M. incognita
Full size table

4 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.