Introduction

Plant parasitic nematodes are among the most devastating pathogens of food, cash and fiber crop, causing an estimated 77 billion dollar crop loses annually and the majority of the losses are caused by root-knot nematodes^1-7.

Root- knot disease reduced plant growth, leaf yield and leaf protein content significantly.  The use of effective chemical pesticides causes the problem of residual toxicity in the treated plants, which results in reduced palatability of the leaves to the feeding silkworm larvae, reduction in growth of the larvae and also in silk production. A number of effective chemical pesticides have been extensively used by farmers^7-12.  An indiscriminate use of plant resources for nematode control has also created a problem for bio-diversity conservation¹³.

To overcome this situation, it has been observed in previous experiments that the use of Cina on mulberry reduced root-knot disease and enriched the sericulture industry^{7,11,12,14,15}.  The crude 90% ethanolic extract of the flowering meristems of  A. nilagirica (1mg/ml concentration) and its potency are marketed by homeopathic pharmacists as Cina^7,11,14.

Here, in this field and silkworms rearing trial, we are trying:

• · To further confirm the effects of Cina-soaked-globules on root-knot disease caused by Meloidogyne incognita (Kofoid & White) Chitwood of mulberry in the naturally infected field condition.
  • · To find out the effects of leaves of Cina treated  infected plants on the leaf consumption and growth of silkworm larvae (Bombyx mori L.),
• · To find the effective rate of rearing (ERR%) [in the rearing house] which directly increases silk production for commercial purposes.

Materials and Methods

Design of the study

The following events were considered during field trial and silkworm rearing:

· Site selection for the field experiment

· Estimation of the nematode population for field trial

· Estimation of total area of root-knot infected field

• · Demarcation for experimental field trial
• · Division of experimental field within the demarcated area
• · Preparation of demarcated experimental field
• · Replication of experimental field
• · Selection and collection of mulberry
• · Planting of mulberry cutting
• · Estimation of growth period of planted mulberry cuttings before treatment
• · Division of plant groups and plots of the demarcated experimental field
• · Division of different batches
• · Pruning of mulberry
• · Preparation of mother tincture for treatments
• · Preparation of potentized drug, Cina
• · Preparation of control medicated globules (without Cina)
• · Preparation of test and control solutions before treatments
• · Mortality test
• · Treatments
• · Analysis of residues
• · Assesment of root-knot diseases
• · Statistical analysis for experimental field trials
• · Hatching and brushing 1^st stage silk worm larvae
• · Division of batches for silkworm rearing
• · Daily record of food supply, larval weight and others observations
• · Harvesting of cocoons from rearing house
• · Statistical analysis for rearing trials

· Estimation of the nematode population before treatment

· Preparation of medicated Cina globules

· Site selection for experimental rearing trial

· Collection of seed for silkworm rearing

A map or plan giving details of the experimental field trial

Site of the experimental plots

The field experiment was carried out at the Sriniketan Sericultural

Composite Unit, Government of West Bengal, India where temperature was

28 + 5°C and relative humidity was 75 + 5%. During experimental trials,

there were same climatic conditions on the field.

Estimation of the nematode population

• · Soil and root samples^4,11,14-17 were taken at random from a mulberry firm spreading over an area of 5.6 acre of land with a view to determine the extent and intensity of Meloidogyne incognita (Kofoid & White) Chitwood infestation at the beginning of the experiments in order to start the treatments with the same population levels.
• · The nematode infected experimental field (0.04acre) was divided into two areas; one was infected untreated with Cina (Control) and other was infected treated with Cina. This root-knot infected experimental field was replicated thrice in space (within the demarcated area)^5-7,10,11 and hence, total area of  naturally nematode infected and demarcated experimental field for trials, were 0.16 acre. During experimental trials, there were the same climatic conditions on the field.

Preparation of demarcated experimental field

Both the naturally infected fields were prepared by removing weeds, irrigating water and interchanging among the demarcated experimental field soils for uniform distribution of manure and nematodes^2-11,17.  It was already mentioned that both the naturally infected experimental fields were replicated thrice within the demarcated land space (covering total area 0.16 acre). It was estimated by regular soil sampling^2-11,17. The experimental field trials were randomized in the infested field by using a completely randomized block design within demarcated area.

Method of sample collection

The experimental field trials were randomized in the infested field by using a completely randomized block design within demarcated area. Regular sampling of soil (at least 40 random sampling; 200g of soil in each sample collected by making a hole of 1.8 cm wide and 6 cm deep) was done to assess the M. incognita population for confirming more or less uniform distribution of manure and nematodes^4,7,18-21.

Plantation of mulberry cutting

Mature three years old mulberry cuttings, Morus alba L. , Cv.’S₁‘(average 25cm length and 20g fresh weight) were collected from the same sericulture field and planted with a gap of 45 cm throughout the experimental fields where there were no difference in soil and there were the same climatic conditions on the experimental field. The planted mulberry cuttings were allowed to grow for a period of three months. Regular rhizospheric soil and root sampling (at random) were done for estimation of nematode population during this three months growth period of mulberry in both the fields^3,5,6,15,-16.  At least 80 random rhizospheric soil samplings  ( 200g in each sample ) were collected from rhizospheric root soil area of root ( 10-15cm X 10-15cm ) and at least 40 random root samplings ( 2g fresh root in each sample ) were collected from newly formed roots (or gall roots ) for determining the intensity or presence of nematodes in both the experimental fields as well as replica also^5,6,11,17,18.

Estimation growth period and nematodes population

After three months growth of mulberry,  M. incognita population was estimated in the rhizospheric soil^16,17 as well as roots^6-10,15-17 (at least 40 random samplings in each area) of mulberry plants in each areas of experimental mulberry field. Total demarcated area of root-knot infected experimental fields including replica were 0.16 acre. The M. incognita infected mulberry plants achieved growth of 50-60 cm in height.

Division of groups and plots

All the infected mulberry plants were divided into 16 plots (20 plants / plot), each measuring the area of 2.5^-03 acre.

The mulberry plants were divided into two-plant groups:

· One was infected untreated with Cina (control) plants and

· Another was infected treated with Cina plants.

The plants groups were again divided into different batches (4-plots / batch and 20 plants / plot).

The batches were:

· Infected untreated with Cina (Control),

· Infected CinaMT treated,

· Infected Cina 200C treated and

· Infected Cina1000C treated.

At first all the plants were pruned, manured with NPK and irrigated every 7 days. Before starting treatment with Cina, rhizospheric soil was interchanged among the plants within the demarcated experimental field  to keep the nematode infestation as uniform as possible in the naturally infected field^4-11. After pruning, the plants were allowed to grow for a period of 104 days when their root-knot disease was assessed. Both the naturally-infected- experimental field trials were replicated three times in the demarcated space and there was a relation to previous one (covering total area 0.16 acre).

Root-knot disease

Rhizospheric soil and root sample were taken at random from all the 16 plots of each plant group that consisted of infected plants including replicas. Meloidogyne incognita populations (10 samples / plot in each plant group) were estimated in the rhizospheric soil^15,16 as well as roots^{2-11,16,17,20} of infected mulberry plants. Total number and surface area of leaves of all plant groups were counted^11,14. Total number of root-galls / plant were counted in the fresh roots of mulberry plants^4-7,17-20. The total protein content (fresh) of the leaf and root samples (10 at random sampling / plot) from the 16 plots of each plant group including replicas, were determined^18-20. All the data from all experiments were compared for statistical analysis by analysis of variance (ANOVA). In this field trial, sacrifices of mulberry plants were not done due to well reported  pathological characters from our previous experiments ^4-11,14,15.

Preparation of homeopathic mother tincture

Air-dried and powdered flowering meristems of Artemisia nilagirica (Clarke) pamp were extracted with 90% ethanol at room temperature (25 + 2°C) for 15 days and were filtered for collecting extract. Later, the ethanol from the extract was removed by evaporation at room temperature (25 + 2°C). The residue was dried in a dessicator over anhydrous calcium chloride. The crude residue was dissolved in 90% ethanol at 1mg/ml concentration and was formed into homeopathic mother tincture of A. nilagirica called CinaMT (Original solution or crude extract)^7,11,14_.

Preparation of potentized drug

• · The homeopathic mother tincture of A. nilagirica, (Cina MT) was diluted

with 90% ethanol (1:100) proportionate in a round vial^7,11,14.

• · The vial was filled up to two-third of its space, tightly corked.
• · 10 powerful down ward strokes of the arm were then given.

This process of mechanical agitation is called succession. This is the 1^st centesimal potency marketed by the homeopathic pharmacist under the name of Cina 1C. All the subsequent potencies were prepared by further diluting each potencies with 90% ethanol in the same proportion (1:100) and the mixture was given 10 powerful down ward strokes^{7,11,14,21,22}.  In this way potencies up to Cina 200C and Cina 1000 C were prepared.

Preparation of medicated Cina globules

• · Cina homeopathic potencies in liquid form can be kept in globules. A vial is filled up to two-thirds of its empty space with sucrose globules of a particular size.
• · A few drops of a liquid potency of Cina are poured into the vial to just moisten all the globules.
• · The vial is corked and then shaken so that all globules are uniformly moistened.
• · The cork is 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 is turned upright, well corked and kept in a cool dry place away from light.
• · The dry globules were then kept in a vial. Medicated globules are known to retain their properties for many years^7,11,21,22.  In this process the drug soaked globules, Cina MT, Cina 200C and Cina1000C were prepared.
• · A vial is filled up to two-third of its empty space with sucrose globules of a particular size.
• · A few drops of 90% ethanol are poured in to the vial to just moisten all the globules.
• · The vial is corked and then shaken so that all globules are uniformly moistened.
• · The cork is 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 is turned upright, well corked and kept in a cool dry place away from light.
• · The dry globules were then kept in a vial to retain their properties for many years^7,11,21,22.

Preparation of control globules

In this process the 90% ethanol soaked control sucrose globules were prepared^7,11,21,22. The control globules were prepared in the same way for comparison to the preparation of medicated Cina globules which were prepared with the 90% ethanol.

Preparation of (Cina-) test solutions

The drug soaked globules of Cina MT,Cina 200C and Cina 1000C were then mixed with sterile distilled water in the proportion of 7.2 mg globules / ml of water^7,11,21,22.

Preparation of control solution

The 90% ethanol soaked globules were then mixed with sterile distilled water in the proportion of 7.2 mg globules/ml of water and the control solution was prepared for comparison to the preparation of test solutions^7,11,21,22.

Mortality test

Four sets of cavity block with 1ml distilled water containing 50 larvae (J2) of M. incognita were taken; one set was treated as control and other three were treated as treatment set. To assess the direct effect of Cina test solution, the water was removed by pipette from all the treatment sets, and immediately replaced by 1ml of test solutions: Cina MT, Cina 200C and Cina 1000C (7.2 mg globules/ml concentration) were added respectively. To assess the direct effect of control solution, the control set received 1 ml of control solution and was observed  at 30 minute intervals for a period of 12 hours exposure period at room temperature (25±2°C)^{6,11,14,16,23}. This mortality test was replicated five times. It was noted that both the control (without Cina) and treatment (with Cina) set received sucrose globules^7,11,21,22.

Treatment

· Forty five days after pruning of mulberry plants, all the treatments were done by foliar spray @ 10ml/plant (7.2mg/ml concentration) once daily for 15 days with Cina- test solutions (Cina MT, Cina 200C & Cina 1000C) and control solution respectively.

• · Treatments were given in such a way that all the leaves of the plants were completely sprayed with solutions. During spraying, the soil surface underneath each plant was covered with polyethylene sheet^{4,6,7,11,12,14,15,21}. All the CinaMT, Cina 200C and Cina 1000C treated groups received 10ml/plant test solutions (7.2 mg Cina globules/ml concentration) respectively.
• · The infected untreated with Cina (control) groups were similarly treated 10ml/plant control solutions (7.2mg- 90% ethanol soaked globules/ml concentration) ^{4,7,11,12,14,15,22} .
• · It is noted that the infected untreated with Cina (controls), were not untreated, but treated with the solution made from sugar pills soaked in the alcohol medium. The infected untreated (control) was only treated with the solutions made from sugar globules in the alcohol medium (i.e. without medicine Cina ). At forty four days after the last treatment all the parameters of growth and nematode infection were recorded for each group.

Analysis of residue

A thin layer chromatography plate (TLC) was made with silica gel²⁴. Mulberry leaves, collected one day after the last treatment were homogenized in a blender and extracted with ethanol. The residue was applied at one end of the plate as a small circular spot. The initial spot should be compact for reproducible R_f values and zones should always be placed at the same distance from the surface of developer²⁴. Here, the residues run in thin layer chromatography plate (TLC) with the standard from the Cina- test substances. The test substances were Cina MT, Cina 200C and Cina 1000C^{7,10,11,14, 24}.

Site selection for experimental rearing trial

The site for rearing house was Sriniketan Sericultural Composite Unit, Government of West Bengal, India (with 27±2°C and 70 + 5% RH) ^4-12,25.

Collection seed for silkworm rearing

The eggs or seeds of a mother moth of the multivoltine ‘Nistari’ race(Bombyx

mori L.) were collected from “Regional Sericultural Research and Training Institute, Berhampore-742101, India” ^4-12,25.

Rearing of silkworms

Hatching and brushing 1^st stage silk worm larvae

In the rearing house, silk worm larvae were hatched and hatching rate was 91%.  We then brushed the 1^st stage silk worm larvae in the rearing tray^4-12,25. The larvae were kept inside the rearing chamber of the rearing house at 27±2°C and 70 + 5% RH.

Division of batches for silkworm rearing

The larvae were divided into 4- batches (120 number / batch) and reared ^{5-11,14,15,25}.

The larvae of:

· Batch-I were fed with the leaves of infected untreated with Cina (control) plants,

· Batch-II with the leaves of infected CinaMT treated plants,

· Batch-III with the leaves of infected, Cina 200C treated plants and

· Batch-IV with the leaves of infected Cina 1000C treated plants.

Daily record of food supply, larval weight, harvesting of cocoons and others observations  -

Fresh leaves were given to the larvae 4 times daily. Mulberry leaves were used for feeding one day after the last treatment with Cina. The fresh weight of the larvae and that of the leaves served were recorded daily for each batch until the larvae started spinning. The consumption of fresh leaves [ ( Fresh leaves served - Dry leaves residues -  Fresh leaves initially consumed ) X Moisture loss ], number of feeding and number of feeding day to cocoon formation, number of escaping feeding during moulting, moulting span days and mortality rate were recorded. The fresh silk gland weight ( before start of spinning ), starting time to spinning, span of spinning, harvesting of cocoons, fresh cocoon weight, fresh shell weight, silk layer ratio (Shell weight / Cocoon weight X 100 ) i.e. SR% and effective rate of rearing ( Number of cocoon harvested / Number of silk worm hatched X 100 ) i.e. ERR% were determined ^{7,10,11,14,15,25}. For statistical analysis by ANOVA, ten mature 5^th instar larvae for fresh silk gland weight and ten cocoons for shell (weight fresh) were dissected out in each of the treatment batches including replica of all treatment batches^5-11,25.

Statistical analysis

Here, all the data from experimental field trial and silkworm rearing were used for statistical analysis by one way analysis of variance (ANOVA, Duncan’s multiple range test) with the help of bio-statistic expert²⁶.

Single factor analysis of variance follows the following steps:

· Crude sum of square for total- CSS_total.

· Correction factor- C.F.

· Sum of Square- SS _total.

· Crude sum of square for treatment- CSS_treatment.

· Sum of square for treatment- SS_treatment.

· SS_{(Error or random variation}⁾ =SS_(total) – SS_(treatment) = S_T² – S_V² = S_E^2.

· Follow the ANOVA  F- Table.

· Calculated F-ratio.

· Critical Difference-C.D.

· If difference between any two treatment means is less than C.D., then the

said two treatments are equivalent, and if greater they are not equivalent. All the data from field and rearing trial compare with or at the latest have significant difference level of P<0.01 and P<0.05 from ANOVA  table respectively.