Management of Root-Knot Nematode Meloidogyne javanica through Homeopathic Medicines

Asma Hanif* and Shahnaz Dawar

Department of Botany, University of Karachi, Karachi-75270, Pakistan.

Received | July 23, 2021; Accepted | November 26, 2021; Published | December 07, 2021

*Correspondence | Asma Hanif, Department of Botany, University of Karachi, Karachi-75270, Pakistan; Email: [email protected]

Citation | Hanif, A. and Dawar, S., 2021. Management of root-knot nematode Meloidogyne javanica through homeopathic medicines. Pakistan Journal of Nematology, 39(2): 73-81.

DOI | https://dx.doi.org/10.17582/journal.pjn/2021.39.2.73.81

Keywords | Homeopathic medicines, Plant parasitic nematode, Vitro and vivo experiments

Introduction

Plant parasitic nematodes are major concern for crop production throughout the world (Askary and Haider, 2010; Abd-Elgawad and Askary, 2015). They reduce the quality and quantity of the crop and on an average worldwide crop loss of 12.6% ($215.77 billion), due to these nematodes for only the top 20 life-sustaining crops based on the 2010–2013 production figures and prices has been estimated (Abd-Elgawad and Askary, 2015). Moreover, 14.45% ($142.47 billion) was an average annual yield loss in the subsequent group of food or export crops. Among the plant parasitic nematodes, root-knot nematode, Meloidogyne species are serious pathogen of several crops of agricultural importance (Ali and Askary, 2001; Jones et al., 2013).

In Pakistan, Meloidogyne spp. infect wide range of host plants resulting in heavy economic losses (Zaki, 2000). Infectious second-stage juveniles (J2) enters root tip of susceptible host produces strong interactive relationship with the host root and migrate intercellularly towards the cortex and inhabitant in the vascular bundles (Caillaud et al., 2008) where they establish permanent feeding sites and produce J3 and J4 stages. They made multiple multinucleate giant cells due to hypertrophy and hyperplasia that can be easily distinguished known as “knots or galls” on the roots (Ibrahim et al., 2006; Askary, 2017). Due to the formation of galls, it caused blockage of xylem tissue, reduced the formation of nodules, interferes in nitrogen fixation and disrupt the photosynthetic activity of plants (Abdollahi and Ghazalbash, 2012). RKNs begin the parasitic process by secreting effector proteins through the stylet in the roots of host plant cells (Quentin et al., 2013) which are essential for generating and continuing the feeding site (Williamson and Hussey, 1996). These secreted proteins affect the development, cell cycle, nitrogen degradation and disrupt the physiological and biochemical processes in host cell (Davis et al., 2004; Akker and Birch, 2016).

Fastest management strategy of controlling Meloidogyne spp. in modern era usually achieved by the application of synthetic nematicides (Medina-Canales et al., 2019); however due to toxicity and hazardous produce in the soil ecosystem (Kim et al., 2018) replacement of chemicals by friendly measures have been developed, although pathologist have not been completely successful in attaining the similar levels of effectiveness (Desaeger et al., 2017) which is imperative to note that results of in vivo and in vitro experiments from the similar treatment may differ due to additional factors such as soil pH, moisture content, degradation of nematicide active ingredients, soil structure, temperature or interaction with microorganisms (Xiang et al., 2018; Bell et al., 2019; Sikder and Vestergård, 2020). Therefore, present research carried out with the aim to explore the nematicidal effectiveness of homeopathic medicines in the management of Meloidogyne javanica under vitro and vivo conditions.

Materials and Methods

Extraction of eggs and juveniles

Infected egg plant roots showing characteristic of galls symptom due to M. javanica were obtained from the green house maintained at the Department of Botany were washed thoroughly with sterilized distilled water for twice and cut into small pieces and was put in a Nalgene wide mouth bottle in which three drops of sodium hypochlorite (1.0%) solution was added and placed in mechanical shaker for half an hour. The contents were poured into a 100-mesh sieve, fitted over a 400-mesh sieve were washed under running tap water (2 mins.). Residue obtained on a 400-mesh sieve was transferred into a beaker. Number of eggs and juveniles/ mL suspension was recorded in a counting dish (Hussey and Barker, 1973).

Nematicidal activity of homeopathic drugs using in vitro test

Using both hatching and mortality test of nematodes, Dr. Willmar Schwabe homeopathic drugs of mother tincture (30Q) includes; Abroma augusta, Arnica montana, Artemisia vulgaris, Bellis perennis, Berberis vulgaris, Bryonia alba, Calendula officinalis, Calotropis gigantea, Clerodendron, Cina, Fagopyrum esculentum, Foenidulum vulgare, Hamamelis Virginia, Hedera helix, Inula, Jasminum officinale, Lamium album, Nux vomica, Opuntia, Psoralea corylifolia, Rosmarinus officinalis, Ruta graveolens, Salvia officinalis, Sulphur, Thuja occidenatlis, Trifolium repens, Withania somnifera, Yucca filamentos, Santonine-43 and Kent-20 along with 30C and 200C potencies of all the above tested drugs including Abies nigra, Calcarea carbonica, Caltha palustris, Carbo vegetablis, Natrum muriaticum, Opium and Phosphorus were used to detect the potent nematicidal activity against M. javanica (Cayrol et al., 1989).

For hatching and mortality test, one mL egg suspension (20-50 eggs/mL) and one mL hatched J2 suspension (20-45 juveniles/mL) were taken separately, one mL of tested homeopathic drugs (Q, 30C and 200C) with different concentrations (100, 75 and 50% v/v) were poured in cavity blocks having lids, respectively. Cavity block with sterilized distilled water and absolute alcohol taken as control and kept at room temperature. Each treatment was replicated thrice and after 0, 24, 48, 72 and 96 hours of exposure, numbers of hatching larvae and dead juveniles were counted under stereo microscope and expressed as percentage of the total nematode incubated.

Nematicidal activity of homeopathic drugs using in vivo test

Sandy loam soil was used for pot experiment containing sand (76%), clay (9%) and silt (15%) was determined by Gee and Bauder (1986) method having ≥7.2 pH (Brady, 1990) and organic matter present in the soil was 1.2% (Sparks, 1996). Tested seeds were treated with Santonine-43 and Kent-20 at three concentrations (100, 75 and 50% v/v) separately and dried aseptically. Different concentrations of treated seeds and soil drenched (20mL) with both homeopathic drugs separately and five tested seeds were sown in the 300g of soil. Untreated seeds and without drenched soil served as control and each treatment replicated thrice. The whole experiment was conducted on okra, sunflower, mung bean and mash bean crops. Pots were kept under natural sunlight and after seedling emergence ≈2500 J2 of M. javanica were inoculated by making holes to the nearby host roots and uprooted tested plants after eight weeks of nematode inoculums addition and determine growth parameters and root knot nematode infection.

Results of data was estimated by three-way analysis of ANOVA by using Duncan’s multiple range as proposed by Sokal and Rohlf (1995).

Results and Discussion

In hatching test under vitro condition, mother tinctures of A. augusta, B. perennis, B. vulgaris, F. esculentum, H. virginia, Inula, L. album and N. vomica was least effective against M. javanica eggs at 100 and 75% and also minimum controlled of M. javanica juvenile at 48, 72 and 96 hours (P<0.001) was observed, while complete hatching of eggs was observed at 50% concentration and failed to kill the J2 nematode. A. vulgaris, T. repens and Y. filamentosa at 100% concentration produced smallest nematicidal effect at 96 hours but failed at both concentrations both in hatching and mortality tests. When pure concentration (P<0.001) of A. montana, C. officinalis, C. gigantea, Clerodendron, H. helix, J. officinale, P. corylifolia, R. officinalis, R. graveolens, T. occidentalis and W. somnifera were used, they exhibit maximum hatching and mortality of M. javanica at 96 hours. However, mother tincture of Cina resulted in better reduction in hatching followed by 30C and 200C potencies recorded in all three concentrations. Cina (30Q) showed greater effect at 100 and 75% concentrations (P<0.05) which caused the death mortality of juveniles at 96 hours followed by 50%. Using Cina at 200C potency at 100% found greater mortality of juvenile at 96 hours but when 30C potency was used, only 100% showed slightest effect after 72 and 96 hours followed by 75%, while 50% failed in killing the nematode juveniles. When mother tincture of B. alba, F. vulgare, Opuntia and Sulphur were used at different concentrations showed complete hatching and juveniles of M. javanica were fully emerge out after 96 hours indicating no nematicidal activity present in it. Rest of the homeopathic drugs failed in killing the larvae and unable to stop the emerging larvae from the eggs of M. javanica but also the sterilized water and absolute alcohol (control) showed same results. Mother tincture of Kent-20 and Santonine-43 when used at different concentrations gave pronounced effect in reducing the hatching of eggs but also noticed highest mortality of M. javanica after 96 hours (Table 1) selected as the best nematicidal medicine from all the tested drugs. In vitro studies revealed that at 50% concentration, cell free filtrate of all tested bacterial isolates (BT-10, BT-14, BT-16 and BT-64) caused remarkable juveniles’ mortality of Meloidogyne javanica after 24 hours as compared to untreated control (Khan et al., 2010). Many researchers worked on aqueous extracts of medicinal plant leaves proving the nematicidal effect against Meloidogyne sp. by stopping the hatching of egg-masses and enhanced the mortality of juveniles with respect to exposure of time under laboratory conditions (Ibrahim et al., 2006; Dawar et al., 2007; Sultana et al., 2011, Latif et al., 2014; El-Baha, 2017; Neeraj et al., 2017).

In vivo experiment, highest shoot weight and height of mung bean plants showed by Santonine-43 at all concentrations when drenched in soil. Growth parameters such as shoot weight, root length, root weight and number of nodules increased at 100% concentration when Kent-20 was used in both seed treatment and soil drenching methods. However, highest shoot length was recorded at 75% followed by 50% concentration observed in the interaction between the drug, concentration and method. When mother tincture of Santonine-43 drenched in soil it significantly (P<0.001) reduced the galls and egg masses on the roots of mung bean plant followed by Kent-20 (Figure 1). Significant interaction between drugs and concentration on growth parameters (P<0.05) and root knot infection (P<0.001) were noticed. Reduction in the formation of galls and egg masses on mash bean roots significantly (P<0.001) controlled by the mash bean seeds treated with pure Kent-20 followed by 75% concentration. Moreover, both homeopathic drugs at 100% diminished the gall formation when drenched in soil. As compared to control when soil was drenched with 50% concentration of Kent-20 and Santonine-43 drugs respectively showed minimum effect against root knot infection. Significant (P<0.001) effect on shoot/root length and weight was caused by the drug and concentration interaction. 100% of Santonine-43 and Kent-20 showed highest growth parameters on mash bean plants (Figure 2).

 

Table 1: Effective results of homeopathic drugs against M. javanica at different time intervals under vitro condition.

Homeopathic drugs (Mother tincture-30Q)	Conc. (%)	Hatching (%)				Mortality (%)
		Time (Hours)				Time (Hours)
		24±SE	48±SE	72±SE	96±SE	24±SE	48±SE	72±SE	96±SE
Control (Sterilized water)	0	37.3± 0.97	55.3±0.98	79.8±1.25	100±1.44	0.0± 0.0	0.0± 0.0	0.0± 0.0	0.0±0.0
Control (Absolutealcohol)	100	41.2± 2.23	59.6±1.96	80.7±2.16	100±2.41	0.0± 0.0	0.0± 0.0	7.5± 0.47	16.75±1.18
Arnica montana	100	38.9± 1.72	62.2±1.85	77.8±1.69	89.5±1.41	0.0± 0.0	0.0± 0.0	0.0± 0.0	9.62±0.72
Berberis vulgaris	100 75	28.9± 1.24 26.4± 1.84	46.0±1.44 48.9±1.18	60.0±1.25 69.9±1.24	80±1.26 90.3±0.94	0.0± 0.0 0.0± 0.0	0.0± 0.0 0.0± 0.0	0.0± 0.0 0.0± 0.0	9.5±0.71 0.0±0.0
Calendula officinalis	100 75	31.9± 0.98 41.7 ±0.97	57.5±0.98 59.6±1.44	74.4±0.23 76.1±1.44	85.3±0.72 90.4±0.71	0.0± 0.0 0.0± 0.0	12.4± 0.94 0.0± 0.0	25.7± 0.97 12.8± 0.72	37.1±0.47 35.0±0.46
Calotropis gigantea	100	22.4± 0.94	40.3±1.85	55.2±1.38	78.3±1.25	0.0± 0.0	0.0± 0.0	0.0± 0.0	36.0±1.69
Clerodendron	100	36.8± 2.19	61.8±2.49	75.0±1.69	90.4±2.87	0.0± 0.0	0.0± 0.0	0.0± 0.0	14.8±1.18
Cina	100 75 50	17.1± 0.72 23.7± 0.94 18.5± 0.93	49.3±1.44 50.2±0.98 43.3±0.98	64.5±0.46 68.4±0.97 66.7±0.94	76.9±0.27 80.2±0.71 85.2±1.25	8.6± 0.47 0.0± 0.0 0.0± 0.0	17.1± 0.47 18.5± 0.72 0.0± 0.0	23.7± 0.70 26.5± 0.94 34.6± 0.72	42.0±1.78 34.4±0.72 51.3±0.97
Fagopyrum esculentum	100	29.7± 0.98	41.2±0.72	56.8±1.18	77.8±0.47	0.0± 0.0	12.6± 0.46	18.9± 0.46	25.2±0.46
Hamamelis virginia	100	17.9± 1.41	35.1±0.98	53.2±0.72	63.3±0.72	0.0± 0.0	0.0± 0.0	14.4± 0.94	26.3±0.72
Inula	100	28.9± 0.94	44.4±0.94	62.2±0.54	80.7±0.97	0.0± 0.0	1.7± 0.54	6.4± 0.72	11.9±0.71
Jasminum officinale	100	31.0 ±1.69	54.1±1.89	71.4±0.62	83.8±0.97	0.0± 0.0	0.0± 0.0	15.2± 0.47	25.5±1.18
Lamium album	100	19.2± 1.44	44.8±1.44	61.6±0.95	74.1±1.41	0.0± 0.0	0.0± 0.0	0.0± 0.0	19.6±0.72
Psoralea corylifolia	100	42.9± 1.25	59.1±1.08	76.3±0.62	86.6±1.36	0.0± 0.0	13.6± 0.47	28.8± 0.44	49.4±0.44
Rosmarinus officinalis	100	24.9± 1.44	42.6±1.44	58.9±0.42	75.1±0.94	0.0± 0.0	0.0± 0.0	0.0± 0.0	23.1±0.72
Salvia officinalis	100	26.2± 1.18	49.9±1.78	71.6±2.62	87.2±1.24	0.0± 0.0	0.0± 0.0	0.0± 0.0	8.0±0.54
Thuja occidentalis	100	25.3± 1.65	47.5±1.25	68.5±2.12	84.8±1.08	0.0± 0.0	9.3± 0.72	19.8± 0.44	34.8±0.72
Trifolium repens	100	29.9± 1.51	54.1±1.78	73.7±0.98	91.6±0.54	0.0± 0.0	0.0± 0.0	6.3± 0.40	15.7±0.47
Withania somnifera	100 75 50	31.2± 0.97 35.3± 0.94 29.5± 0.94	51.5±1.65 55.4±0.94 53.8±0.98	68.9±0.47 75.6±1.62 79.5±0.94	82.6±0.94 90.7±0.93 91.6±0.72	0.0± 0.0 0.0± 0.0 0.0± 0.0	14.4± 0.94 0.0± 0.0 0.0± 0.0	25.9± 0.41 0.0± 0.0 0.0± 0.0	37.5±0.46 19.4±0.72 0.0±0.0
Kent-20	100 75 50	10.2± 0.98 25.3± 0.72 37.0± 1.25	25.6±1.69 39.9±1.51 53.8±0.47	40.4±1.65 49.6±1.41 61.6±0.72	47.6±0.83 59.2±0.56 74.1±0.54	48.4± 0.72 37.0± 0.71 32.6± 0.97	70.7± 0.47 55.4± 0.45 55.1± 0.96	92.9± 0.97 75.8± 0.42 78.6± 1.18	100±1.95 94.2±1.18 93.6±2.04
Santonine-43	100 75 50	12.0± 2.19 20.2± 0.72 28.9± 0.98	27.6±1.18 31.7±0.47 38.5±0.94	41.5±0.81 41.4±0.94 46.2±0.93	51.3±0.72 50.5±1.18 55.6±1.18	37.7± 0.98 40.3 ±1.24 22.4± 0.94	72.2± 2.59 58.6± 0.94 51.1± 0.27	91.6± 2.04 73.3± 0.72 77.6± 1.44	100±2.12 90.5±1.18 94.9±1.51
Cina (200C)	100 75 50	26.5± 0.94 30.0± 0.72 41.9± 0.71	54.2±0.72 45.5±1.25 64.2±1.41	85.8±0.98 79.6±1.36 86.5±1.51	100±1.18 100±1.44 100±1.43	0.0± 0.0 0.0± 0.0 0.0± 0.0	29.9± 1.18 15.2± 0.98 0.0± 0.0	41.2± 0.72 37.0± 0.47 27.1± 0.72	53.2±0.47 54.7±0.72 45.8±0.73
Cina (30C)	100 75 50	44.3± 0.72 39.4± 0.83 42.8± 0.94	86.7±0.72 75.2±0.97 80.8±0.98	93.8±0.47 88.2±0.97 93.3±0.94	100±0.27 100±0.72 100±1.24	0.0± 0.0 0.0± 0.0 0.0± 0.0	0.0± 0.0 0.0± 0.0 0.0± 0.0	33.9± 0.72 0.0± 0.0 0.0± 0.0	55.9±1.18 12.2±0.72 0.0±0.0
LSD0.05= (C)= 2.549, (P)= 14.865, (T)= 2.944						LSD0.05= (C)= 1.151, (P)= 6.713, (T)= 1.329

 

Where; Conc. /(C): Concentrations; (P): Potency of drugs; (T): Time; SE: Standard Error.

 

Application of pure Santonine 43 was effective in improving sunflower height and weight (P<0.001; P<0.05) followed by 75% concentration. However, root length and weight were amplified due to sunflower seeds treated with Kent-20 at 75 and 50% concentration. Both pure homeopathic drugs (P<0.001) gave noticeable reduction in the galls formation and egg masses recorded in seed treatment and soil drenching methods followed by 75 and 50% concentrations (Figure 3). Better shoot/root length and weight of okra were achieved by Kent-20 when drenched in soil (100%) followed by Santonine-43. Heavy root weight of okra was recorded when 75% concentration of Santonine-43 drug drenched in soil. Okra seeds treated with 100% concentration of both homeopathic drugs decreased the galls and number of egg masses significantly (P<0.001) which results in the improvement of plant growth as compared to control in which occurrence of infection were noticed due to formation of galls produced by M. javanica (Figure 4). The soil-inhabitant nematode can be controlled by a broad range of organic amendments such as biofertilizers, crop residues or byproducts of plants, composts, green or animal manures either applied “in vitro” and “in vivo” assays significantly decreased the population (Hu and Cao, 2008; Hu and Qi, 2010; McSorley, 2011; Mennan and Melakeberhan, 2010; Soheili and Saeedizadeh, 2017). Used of homeopathic drugs in the control of Meloidogyne spp., studied by various researchers proving positive nematicidal effects (Datta, 2006; Carneiro et al., 2010; Carneiro, 2011). Present results showed that out of three concentrations used (100, 75 and 50%), tested seeds treated with 100% of Kent-20 and Santonine-43 found best in the suppressing of galls formation and reduced egg masses remarkably produced healthy crops (sunflower, okra, mung and mash beans) followed by the 75 and 50% concentrations.

 

 

Throughout the world, nematologist have been putting efforts into developing new environmentally benign strategies in the management of Meloidogyne spp. having nematicidal activity such as application of beneficial microbes (bacteria/fungi) essential oils, plant extracts, green manure, oil seeds cake, mulching, use of natural drugs derived from plants active compounds etc. are some of the ecofriendly treatments that have been tested for their efficacy against root knot nematodes (Tiyagi et al., 2002; Irshad et al., 2006; Ogwulumba and Ugwuoke, 2011; Ghazalbash and Abdollahi, 2013; Sivasakthi et al., 2014; Kokalis-Burelle et al., 2016; Askary, 2012, 2020). Recently, homeopathic pellets showed positive results in killing the J2 of Meloidogyne javanica on agricultural field. Soil amended with homeopathic pellets (Kent-20 used at 75% v/w concentration) produced healthy seedlings of okra, sunflower, mung and mash bean as it suppressed the M. javanica infection as compared to control (non-amended with homeopathic pellets) which produced galls on roots and disrupt the growth of tested crops (Hanif and Dawar, 2019). Experimental researches on the principle of homeopathy in plants were mostly performed by Mexico, India, Europe and Brazil recently (Marques et al., 2011).

 

Homeopathic medicines in prescribing substances either highly diluted or potentized form provide potential technology for sustainable agriculture (Rossi et al., 2004) due to controlling plant diseases (El-Mougy et al., 2004) showed inexpensive, eco-friendly and used in little doses (Toledo et al., 2011). Treated plants with homeopathy protect against root knot nematode attack and result in better crop yield (Hanif and Dawar, 2018).

Conclusions and Recommendations

Since vermicide homeopathic drug is still very little studied, there are many divergences about it. Homeopathic drugs (30Q, 200C and 30C potencies) were tested at different concentrations to check the hatching and mortality of M. javanica, all drugs showed negative results except Cina (in all potencies) but not as excellent as compared to Kent-20 and Santonine-43 showed complete mortality of nematode at 96 hours exposure which found to be best vermicide medicine which was further checked in the vivo experiment and confirmed its efficacy on the leguminous and non-leguminous plants by actively reducing galls formation which was recorded in both seed treatment and soil drenching methods. Conclude that both the methods are effective in reducing the infestation of M. javanica on plant root. However, drenching the soil with homeopathic drugs are difficult on large scale in famers’ field. On the other hand, seed treatment is easy and cost-effective method which can be recommended for the crops grown in microplots.

 

Novelty Statement

Seeds treated with homeopathic medicines found highly effective in controlling root knot nematode especially Meloidogyne javanica can be used as eco-friendly method in commercial scale.

Author’s Contribution

Asma Hanif and Shahnaz Dawar: Contributed equally.

Conflict of interest

The authors have declared no conflict of interest.

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