Control of Root-Knot Disease of Mulberry by Homeopathic Medicines: Aakashmoni [Mt, 30c, 200c & 1000c] Prepared from the Funicles of Acacia Auriculiformis

Eco-Club Research Unit, Kanchannagar D.N.Das High School,

Kanchannagar, Burdwan, West Bengal, India and Life Sciences Unit, Burdwan

Model School, Dewandighi, Burdwan.

ABSTRACT

Root-knot disease of mulberry caused by the nematode Meloidogyne incognita (Kofoid & White) Chitwood, has important economic implications for sericulture (silk farming). The Homeopathic medicines; Aakashmoni mother tincture (MT) and potentised Aakashmoni 30C, Aakashmoni 200C and Aakashmoni 1000C, prepared from the crude extract from the funicles of Acacia auriculiformis A. Cunn , will be applied by foliar spray on mulberry (Morus alba L.,cv.S₁) infected with M. incognita juveniles (J2). Pre-treatment (ending 6 days before inoculation) and post-treatment (starting 6 days after inoculation) schedules will be tested. The uninoculated control batches will be treated with the same procedure with Aakashmoni mother tincture (MT), Aakashmoni 30C, Aakashmoni 200C and Aakashmoni 1000C. Both pre- and post-treatment significantly reduced nematode infection in terms of root gall number and nematode population in root. All the treated plants showed improved growth in terms of fresh biomass of shoot and root, length of shoot and root, number of leaves, leaf surface area, root and leaf-protein content. Aakashmoni 200C is the most effective medicine in all respects of nematode control as well as growth of the test plants.

Pre-treatments show slightly better effects than the post-treatments. It is interesting that inoculated and treated plants not only are less affected by nematodes but also have a better growth than uninoculated untreated controls.

Keywords: Root-knot disease, homeopathic medicines, Aakashmoni, Acacia auriculiformis, mulberry, control.
Introduction
Mulberry is an important plant in the economy because silk production depends on the nutritive quality of the leaves, which is hampered by pathogen attack (like nematodes, fungus, virus, bacteria, insects etc). These pathogens are the main obstacles causing considerable loss in yield and nutritive value of mulberry foliage. Feeding of the diseased leaves affects the health of the silkworms adversely and the cocoon yield in terms of quality and quantity. The lack of regular and systematic studies on the occurrence of various diseases and epidemics is responsible for the recurring loss in leaf yield (Powell, 1971; Sengupta et al.1990 ; Teotia and Sen, 1994; Datta et al., 1997; Datta, 1999, 2005a, 2005b, 2006a, 2006b, 2007, 2010; Datta and Datta, 2006a, 2007a, 2007b, 2008). The use of chemical pesticides like Furaden may achieve a measure of control of those mulberry diseases, but there remains the problem of residual toxicity in the treated plants and this toxicity results in reduced palatability of the leaves to the feeding silkworm larvae, reduction in growth of the larvae and also in silk production ( Teotia and Sen, 1994; Paul et al., 1995; Sukul et al., 2001; Datta, 2007, 2010). Root-knot disease of mulberry, caused by the nematode Meloidogyne incognita (Kofoid and White) Chitwood, is very wide spread affecting more than 80% of plantations in parts of India (Teotia and Sen,1994).

A number of plant bio-nematicides though effective and easily biodegradable, are not easily available in large quantities from natural sources, and isolation of only a small quantity of an effective metabolite requires huge quantities of plant materials. This would result in rapid depletion of natural resources, particularly in tropical regions. Indiscriminate use of plant resources has already created problems of biodiversity conservation in these areas (Halfer,1996; Datta,1999, 2005a, 2005b, 2006a, 2006b, 2007, 2010). Bio-nematicides from animal origin (nematode extract) reduce Meloidogyne incognita infestation in different plants and root callus by using their defence- response against M. incognita infection, but it has some problems ( Datta et al., 1997, 1998a, 998b; Datta,1999, 2005a, 2005b, 2006a, 2006b, 2007 ; Datta and Datta, 2006a, 2006b, 2007a, 2007b, 2008). These are the main problems.

The need to overcome this situation has prompted me to prepare the present work with a view to studying the use of potentized homeopathic medicines; Aakashmoni mother tincture (MT), Aakashmoni 30C, Aakashmoni 200C and Aakashmoni 1000C, prepared from the crude extract from the funicles of Acacia auriculiformis A. Cunn will be applied by foliar spray on mulberry (Morus alba L.,cv.S₁) infected with Meloidogyne incognita (Kofoid & White) Chitwood juveniles(J2). Pretreatment (ending 6 days before inoculation) and post-treatment (starting 6 days after inoculation) schedules will be tested. The uninoculated control batches will be treated with the same procedure with Aakashmonimother tincture (MT),Aakashmoni30C,Aakashmoni 200C and Aakashmoni 1000C. After harvesting, plant growth will be assessed in terms of fresh biomass of shoot and root, length of shoot and root, number of leaves, leaf surface area, root and leaf-protein content, and root-knot disease by root gall number and nematode population in root.

The main objectives are:

â€¢ To develop suitable potentized homeopathic medicines; Aakashmoni mother tincture (MT), Aakashmoni 30C, Aakashmoni 200C and Aakashmoni 1000C from the crude extract of the funicles of Acacia auriculiformis A. Cunn. against root-knot nematodes Meloidogyne incognita (Kofoid & White) Chitwood of mulberry plants (Morus alba L., cv. S1) and that medicine will be easily available, non-phytotoxic and non-pollutant as well as conserve our biodiversity.

â€¢ To investigate the effects of Aakashmoni by foliar spray on root-knot disease caused by M. incognita of mulberry.
â€¢ To find out the growth of healthy and infected plants treated with Aakashmoni.

Materials and Methods

Preparation of homeopathic Aakashmoni mother tincture:

Air-dried and powdered funicles of Acacia auriculiformis A. Cunn will be extracted with 90% ethanol at room temperature (25 + 2°C) for 15 days and will be filtered for collecting extract. Later, the ethanol from the extract will be removed by evaporation at room temperature (25 + 2°C). The residue will be dried in a desicator over anhydrous calcium chloride. The crude residue will be dissolved in 90% ethanol at 1mg/ml concentration and will form a homeopathic mother tincture of A. auriculiformis, named Aakashmoni MT (Original solution or crude extract) (Anonymous,1920; Mahato et al., 1992; Datta et al., 1997, 1998b; Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

Preparation of potentized Liquid Aakashmoni medicine:
· The homeopathic mother tincture of A. auriculiformis, named in reference to Tegore Rabindranath Thakur, Aakashmoni MT will be diluted with 90% ethanol (1:100) proportionate in a round vial.

· The vial will be filled up to two-third of its space, tightly corked.

· And then will be given 10 powerful down ward strokes of the arm.

This process of mechanical agitation is called succussion. This is the 1^st centesimal potency named Aakashmoni 1C. All the subsequent potencies will be prepared by further diluting each potency with 90% ethanol in the same proportion (1:100) and the mixture will be given 10 powerful downward strokes. In this way potencies up to Aakashmoni 30C, Aakashmoni 200C and Aakashmoni 1000C will be prepared (Anonymous,1920; Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

See also

Ask the Plant Doctor - January 2010

Preparation of medicated Aakashmoni globules:
· Aakashmoni homeopathic potencies in liquid form can be kept in globules. A vial will be filled up to two-third of its empty space with sucrose globules of a particular size.

·Afew drops of a liquid potency ofAakashmoni will be poured into the vial to just moisten all the globules.

· The vial will be corked and then shaken so that all globules will be uniformly moistened.

· The cork is loosened and the vial is turned upside down to allow excess liquid drain out.

· After keeping the vial in the inverted position for nine to ten hours, the vial will be turned upright, well corked and kept in a cool dry place away from light.

· The dry globules will then be kept in a vial and medicated globules are known to retain their properties for many years. In this process the drug soaked globules; Aakashmoni MT, Aakashmoni30C, Aakashmoni 200C and Aakashmoni 1000C are prepared (Anonymous,1920; Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

Preparation of control globules:
· A vial will be filled up to two-third of its empty space with sucrose globules of a particular size.

· A few drops of 90% ethanol will be poured in to the vial to just moisten all the globules.

· The vial will be corked and then shaken so that all globules are uniformly moistened.

· The cork will be loosened and the vial is turned upside down to allow excess liquid to drain out.

· After keeping the vial in the inverted position for nine to ten hours, the vial will be turned upright, well corked and kept in a cool dry place away from light.

· The dry globules will be then kept in a vial to retain their properties for many years.

In this process the 90% ethanol soaked control sucrose globules will be prepared. The control globules will be prepared in the same way for comparison to the preparation of medicated Aakashmoni globules which will be prepared with the 90% ethanol media (Anonymous,1920; Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

Preparation of Aakashmoni test solutions:

The drug soaked globules of Aakashmoni MT, Aakashmoni 30C, Aakashmoni 200C and Aakashmoni 1000C will then be mixed with sterile distilled water in the proportion of 7.2 mg globules / ml of water (Anonymous,1920; Datta, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

Preparation of Aakashmoni control solution:

The 90% ethanol soaked globules will then be mixed with sterile distilled water in the proportion of 7.2 mg globules/ml of water and the control solution will be prepared for comparison to the preparation of test solutions (Anonymous,1920; Datta, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

Mortality test:

Five sets of cavity block with 1ml distilled water containing 50 larvae (J2) of M. incognita will be taken; one set will be treated as control and other four will be treated as treatment set. To assess the direct effect of Aakashmoni- test solution, the water will be removed by pipette from all the treatment sets, and immediately replaced by 1ml of test solutions: Aakashmoni MT, Aakashmoni 30C, Aakashmoni 200C and Aakashmoni 1000C (7.2 mg globules/ml concentration) will be added respectively. To assess the direct effect of control solution, the control set will be received 1 ml of control solution and observed with every 30 minutes interval for a period of 12 hours exposure period at room temperature (25±2°C). This mortality test will be replicated five times. It will be noted that both the control (without Aakashmoni ) and treatment (with Aakashmoni) set will be received sucrose globules ( Fenner, 1962; Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta et al., 1998b).

Planting and inoculation:

Earthen pots of 22.5 cm diameter and 22.5 cm depth will be divided into eight batches of 20 pots each; each pot contained 12.5 kg of clay soil and compost manure in the proportion of 2:1v/v, and after denematisation of the soil in the pots, they will be planted with a mulberry cutting Morus alba L. cv. ‘S1’ cutting (10 in length and 12 g fresh weightâ€”one mulberry cutting/pot) at day 0. Cuttings will be provided by the Sericultural Composite Unit, Sriniketan, Birbhum, West Bengal, India. The date of planting will be called day-0 (Christie and Perry, 1951; Allen et al., 1983; Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

The groups will be :

1â€¢ uninoculated untreated (control),

2â€¢ uninoculated Aakashmoni MT treated (control),

3â€¢ uninoculated Aakashmoni 30C treated (control)

4â€¢ uninoculated Aakashmoni 200C treated (control),

5â€¢ uninoculated Aakashmoni 1000C treated (control)

6â€¢ inoculated untreated (control),,

7â€¢ inoculated pretreated Aakashmoni MT,

8â€¢ inoculated preteated Aakashmoni 30C,

9â€¢ inoculated preteated Aakashmoni 200C,

10â€¢ inoculated preteated Aakashmoni 1000C,

11â€¢ inoculated post-treated Aakashmoni MT,

12â€¢ inoculated post-treated Aakashmoni 30C,

13â€¢ inoculated post-treated Aakashmoni 200C,

14â€¢ inoculated post-treated Aakashmoni 1000C.

The pots will be denematised by boiling water five times daily for 3 consecutive days before planting. Nine batches of pots would be inoculated at day 34 after the planting of mulberry cutting with

M. incognita J2 larvae @ 2000±55 larvae/pot. The remaining five batches will be served as controls:

â€¢ uninoculated untreated (control),

â€¢ uninoculated Aakashmoni MT treated (control),

â€¢ uninoculated Aakashmoni 30C treated (control)

â€¢ uninoculated Aakashmoni 200C treated (control),

â€¢ uninoculated Aakashmoni 1000C treated (control).

The treatments would be replicated in three batches in each experiment. Nematode population in soil will be monitored 1 month after inoculation, then at 2 weekly intervals. The experiment would be done outdoors in the garden of the Department of Zoology, Visva- Bharati University, Santiniketan-731235, West Bengal, India at an ambient temperature of 30±5^oC and relative humidity 75±5% with irrigation and manuring when necessary(Sengupta et al., 1990. Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

Treatment:

All treatments will be given by foliar spray @of 10 ml/plant four times at an interval of 3 days. The drugs (test solution) and control solutions will be sprayed on leaves in five instalments at intervals of 2.5 h during every treatment day. Treatments will be done; either before inoculation with M. incognita juveniles (pretreated batches) or without inoculation (uninoculated control batches). All the pretreatments would be started at day-15, ending 6 days before inoculation of M. incognita at day-27. The treatments of the post-treated batches would be started 6 days after inoculation of nematodes on day-41.All the Aakashmoni MT treated control batches will be given as foliar spray with Aakashmoni @ 10 ml/plant. The uninoculated untreated (control) and inoculated untreated batches will be treated with control solutions by foliar spray @ 10 ml/plant. During spraying, the soil surface underneath each plant will be covered with polyethylene sheet( Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

See also

Plant Doctor – Oct 2017

Harvesting:

Sixty days after the last treatment (d 144) of the post-treated batches, all the plants would be uprooted and the following parameters would be recorded; shoot length and fresh weight, root length and fresh weight, number of leaves/plant, surface area of leaves, number of root galls/plant, nematode population in root (2 g) and soil (200 g) and leaf and root-protein content. For protein estimation, three samples of leaves and roots would be taken at random from each batch of plants and the total protein fraction in each sample will be estimated by the Folin-Phenol method. All the data would be analysed by one way analysis of variance (ANOVA) (Lowry et al., 1951; Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).

Analysis of residues:

Mulberry leaves, collected 3 days after the last treatments, would be homogenised in a blender and would be extracted with ethanol. The residue will be run in a TLC plate with the standard of the test substance. The test substances will be Aakashmoni MT, Aakashmoni 30C, Aakashmoni 200C and Aakashmoni 1000C( Datta, 1999, 2005a, 2006a, 2008, 2009, 2010; Datta and Datta, 2008; Sukul et al., 2001).
Work plan:
The following events would be considered during pot experiment:
· Site selection for the experiment,
· Estimation of the nematode population for trial,
· Division of experimental pots,
· Replication of experiment,

· Selection and collection of mulberry,

· Plantation of mulberry cutting,

· Estimation growth period of planted-mulberry-cuttings before treatment,
· Estimation of the nematode population before treatment,
· Division of plant groups,

· Division of different batches,

· Pruning of mulberry,

· Preparation of mother tincture for treatments,

· Preparation of potentized homeopathic medicines Aakashmoni,
· Preparation of medicated Aakashmoni globules,
· Preparation of control medicated globules (without Aakashmoni ),

· Preparation of test and control solutions before treatments,

· Mortality test,

· Treatments,

· Analysis of residues,

· Assesment of root-knot diseases,

· Statistical analysis for experimental trials.

Schedule

After the preparation and denematisation of the soil filled pots, the following events occurred:

Day 0: planting of mature mulberry cutting in the denematised soil-filled pots (onecutting/pot).

Day 30: Pruning of mulberry (15 day before pre-treatment).

Day 46, 49, 53 and 57: four pretreatments (pretreatment groups) with Aakashmoni, ending 6 days before inoculation with M. incognita juveniles (J2).

Day 64: inoculation of M. incognita J2 larvae @ 2000±55 larvae/pot.

Day 71, 75, 79 and 83: four post-treatments (post-treatment groups) with Aakashmoni and starting 6 days after inoculation of M. incognita juveniles (J2).

Day 87: collection of mulberry leaves for thin layer chromatography (TLC).

Day 0 to 143: plant growth period.

Day 144: harvesting of all plants 60 days after the last treatment of inoculated Post-treated Aakashmoni batches.

Results

The effects of treatment with the test solutions Aakashmoni (MT, 30C, 200C and 1000C) on root-knot nematode infection of mulberry are given in Tables 1. Aakashmoni treated plants showed higher protein content in leaves and roots (P ? 0.01, ANOVA). Nematode population and root gall number were significantly lower in the roots of plants treated with the test substances Aakashmoni (MT, 30C, 200C and 1000C) than in the untreated plants (P? 0.01, ANOVA). Nematode populations in the rhizospheric soil in all Aakashmoni-treated batches were significantly higher than in the untreated groups whilst the population in the roots were significantly lower in the treated groups compared to the untreated groups (P ? 0.01, ANOVA). Number of leaves and leaf surface area were also significantly higher in all Aakashmoni treated plants than in both the inoculated untreated and uninoculated control plants (P ? 0.01, ANOVA). Nematode population in all the Aakashmoni- treated batches were significantly higher in rhizospheric soil than in root (P ? 0.01, ANOVA). Aakashmoni had no direct effect on nematode mortality within the exposure period of 12 h and Aakashmoni had left no toxic residues of the test substance in the treated mulberry plants, collected 3 days after last treatment, by TLC. This experiment was repeated thrice with similar results, all the data are reproduced here (Tables 1).

Discussion

It has been already observed that the extract from the funicles of Acacia auriculiformis A. Cunn. and its pure compounds acaciasides (A & B), are highly effective in reducing mulberry diseases like root-knot, fungus, virus, bacteria and tukra leaving no residual toxicity in the leaves to affect the growing silkworm larvae (Datta et al., 1997, 1998a, 998b; Datta,1999, 2005a, 2005b, 2006a, 2006b, 2007; Datta and Datta, 2006a, 2006b, 2007a, 2007b, 2008). However, Aakashmoni [30C, 200C and 1000C] is too dilute to contain any drug molecules (Anonymous,1920; Datta and Datta, 2006a, 2006b, 2007a, 2007b, 2008; Datta,1999, 2005a, 2005b, 2006a, 2006b, 2007, 2010). It is surprising that inoculated and treated plants not only are less affected by nematodes but also have a better growth in all respects than uninoculated controls. It is also interesting that Aakashmoni treatment effects were the most pronounced of all Aakashmoni medicines. Pre-treatment was generally more effective than post-treatment. The effective compounds did not affect the nematodes directly, and mulberry leaves did not contain any residues of the test substance. Aakashmoni not only reduced root-knot disease significantly but also improved the nutritive value of leaves. This study shows that Aakashmoni homoeopathic treatments stimulate resistance against the nematode-pathogen and that the pre-treatments are more effective than the post-treatments. Large numbers of M.incognita larvae were present in the rhizospheric soil in all Aakashmoni treated plants. It may be that Aakashmoni can induce synthesis of some antagonistic substances in the treated plants (Datta et al., 1997, 1998a, 998b; Datta,1999, 2005a, 2005b, 2006a, 2006b, 2007, 2008, 2010; Datta and Datta, 2006a, 2006b, 2007a, 2007b, 2008; Nandi et al., 2003; McClure et al., 1973). M.incognita is known to share common antigens with its host plants (McClure et al.,1973). It appears that during natural infection with the nematode, the host plant shows minimal defense responses to the nematodes because of this antigenic similarity. Aakashmoni medicines may induce natural defense responses in plants against nematode parasites.

Conclusions

The potentised homoeopathic drug Aakashmoni at an ultra high dilution is effective against root-knot disease and improves the growth of host plants. The higher nutritive value of the treated plants contributes to higher growth of larvae and silk production. These results suggest that plant diseases may be effectively controlled by the potentised drug without disturbing the biosphere and we can conserve our biodiversity with cost effective potentised homoeopathic medicines. Further research should be conducted.