Full name of the author’s institutional affiliation :

Subhas Chandra Datta, Ph.D.

Department of Zoology,Visva-Bharati University, Santiniketan-731235, and Life Science Unit, Ajodhya High School, Bankati, Burdwan-713148, West Bengal, India.

Rupa Datta(Nag), M.Sc.

Department of Zoology,Visva-Bharati University, Santiniketan-731235, and  Burdwan Model School, Dewandighi, Katwa Road, Burdwan-713101, West Bengal, India.

Keywords : Potentized Artemisia nilagirica Extract (Cina); mulberry; root-knot; Silk; Effective rate of rearing; field trial.

Running head : “Potentized Artemisia nilagirica Extract increase…..”.

Correspondence by conventional mail : Dr. Subhas Chandra Datta, Researcher & Assistant teacher., C/O- Rajendranath Nag, Bajeprotappur (Katwa Road), Burdwan-713101,West Bengal, India.

Phone No. : 0342-2622097(Res) & 0343-2643225(Off). Cell No. : 09832267610 & 09832192464. Fax No. : 0091-342-2624263 begin_of_the_skype_highlighting              0091-342-2624263      end_of_the_skype_highlighting begin_of_the_skype_highlighting              0091-342-2624263      end_of_the_skype_highlighting.

E-mail : dattasubhas@rediffmail.com

Abstract.

Objectives :  Here, we show the effects of Potentized Artemisia nilagirica Extract (Cina) soaked globules on root-knot disease caused by Meloidogyne incognita (Kofoid & White) Chitwood of mulberry (Morus alba L. , Cv. ‘S₁‘)  in the naturally infected field condition, and also to examine the growth of healthy and infected plants treated with Cina, and lastly examine the effects of leaves of Cina-treated healthy and infected-plants on the leaf consumption and growth of silkworm larvae (Bombyx mori L.) and effective rate of rearing (ERR%).

Design : In this field trial, sucrose globules, soaked with CinaMT, Cina 200C and Cina 1000C respectively, were mixed with distilled water @ 7.2 mg/ml and applied by foliar spray @ 10ml/plant on healthy and M. incognita infected mulberry plants once daily for 15 days.

Results : All Potentized Artemisia nilagirica Extract (Cina) treated healthy and infected plants showed improved growth in terms of number and surface area of leaves, and protein content (%) of leaf and root. Healthy plants treated with Cina 200C shows the greatest positive growth. All nematode infected plants treated with Cina had significantly reduced root-knot disease in terms of root gall number and nematode population in root and soil. No toxic residues in leaves were traced by Thin Layer Chromatography (TLC) one day after the last treatment. Silkworm larvae feeding on the leaves of Cina-treated plants showed improved growth, increased silk gland weight, shell weight, shell ratio (SR%) and effective rate of rearing (ERR %), fewer feeding and number of feeding days, shorter starting time of spinning day and span of spinning, shortermoulting time to cocoon formation, and zero mortality rate.

Conclusion :In this field trial, silk worms reared commercially increased silk production and effective rate of rearing (ERR%) by reducing root-knot disease of mulberry without disturbing the biosphere.

Keywords : Potentized Artemisia nilagirica Extract (Cina); mulberry; root-knot; Silk;Effective rate of rearing; field trial

Text.

Introduction

Plant parasitic nematodes are among the most devastating pathogen of food, cash and fiber crop, causing an estimated 77 billion dollar crop loses annually and the majority of the losses is caused by root-knot nematodes^1,2,3. Root- knot disease reduced plant growth leaf yield and leaf protein content significantly and the use of effective chemical pesticides cause 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 the farmers^4,5,6,7. Indiscriminate use of plant resources for nematode control has also created problem for bio-diversity conservation⁸. To overcome this situation, it has been observed in the previous pot experiments, that the use of Cina on mulberry reduced root-knot disease and enriched sericulture industry^7,9. 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^7,9. Here, in this field trial and silkworm rearing, we 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, and also to find out the growth of healthy and infected plants treated with Cina, and lastly to find out the effects of leaves of Cina-treated healthy and infected-plants on the leaf consumption, growth of silkworm larvae (Bombyx mori L.) and effective rate of rearing (ERR%) which directly increases silk production for commercial purpose.

Materials and Methods.

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 relativehumidity was 75 + 5%.

Estimation of the nematode population

Soil and root samples^10,11,12,13 were taken at random from a high bushy mulberry plantation spreading over an area of 5.6 acre of land with a view to determining the extent and intensity of Meloidogyne incognita (Kofoid & White) Chitwood infestation. Later, two separate areas (in the same locality and climatic condition); one concrete soil-filled  land and other naturally root-knot infected land, each measuring 0.02 ha, were demarcated in the mulberry field where there were no soil differences as well as environmental factor.

Preparation of healthy area

The concrete 0.02 ha area (18889.76 x 1066.80 x 45.72 cm³ ) was filled with a mixture of sandy soil, collected from very less root-knot infected soil of the same sericulture land, and yard manure ( 2 : 1 vol / vol ). The soil-filled concrete area was then denematized by continuous flow of boiling water throughout the day for 25 days and never water leached out the soil. Every day, at least 40 random sampling of moist-soil ( 200g of soil i.e., each sample collected by making a hole of 1.8 cm wide and 6 cm deep ) were done in the concrete soil-filled area for 30 days and were assessed for M. incognita population to confirm denematization of soil ^3,11,12. This was a very troublesome process for preparing M. incognita-free soil and there was no scope to replicate this healthy nematode free soil-filled area for field trial.

Preparation of naturally infected area

The other 0.02ha naturally M. incognita infected sandy soil field was prepared by mixing yard manure ( 2 : 1 vol / vol ), removing weeds, irrigating water and interchanging among the soil for uniform distribution of manure and nematodes in the naturally infected field which was estimated by regular soil sampling like a same process of healthy soil-filled area^3,11,12. This naturally infected soil-filled area was replicated thrice.

Plantation of mulberry cutting

Mature three years old mulberry cuttings, Morus alba L. , Cv.’S₁‘(average 25cm length and 20g fresh weight) collected from the same sericulture field, were planted with a gap of 45cm throughout the  experimental  fields where there were no soil differences and climatic conditions. 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 month growth period of mulberry in all fields^3,11,12,15. Atleast 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 all the experimental fields.

Division of groups and plots

After three months growth of mulberry,  M. incognita population were estimated in the rhizospheric soil^11,12 as well as roots^5,11,12,13 (at least 40 at random sampling in each area ) of mulberry plants in each areas of mulberry field. A total area of 0.04 ha was divided into two main fields separately; one was a healthy mulberry plant’s field ( concrete soil-filled area) and the other was a naturally infected mulberry plants field, each measuring 0.02 ha. The healthy and M. incognita infected mulberry plants achieved growth of  50-60 cm in height. The healthy and infected mulberry plants were divided into 16 plots, each measuring the area of 472.44cm X 533.4cm X 45.72cm. The mulberry plants divided into 8-plant groups (4groups from each healthy plants and another 4 groups from infected plants) and each group has two plots (20 plants/plot). The plant groups were: 1. Healthy (Control),  2. Healthy CinaMT treated, 3. Healthy Cina 200C treated, 4. Healthy Cina 1000C treated, 5. Infected (Control), 6. Infected CinaMT treated, 7. Infected Cina 200C treated and 8. Infected Cina 1000C treated. At first all the plants were pruned, manured with NPK and irrigated every 7 days. Rhizospheric soil was interchanged among the plants to keep the nematode infestation as uniform as possible in the naturally infected field. After pruning, the plants were allowed to grow for a period of 104 days when their root-knot disease was assessed. The field trial was replicated three times, except for the healthy plant group.

Root-knot disease

Rhizospheric soil and root sample were taken at random from all the 8 infected plots. Meloidogyne incognita populations (10 samples / plot in each plant group) were estimated in the rhizospheric soil^11,12 as well as roots^{5,11,12,13,15} of infected mulberry plants. Total number and surface area of leaves of all plant groups were counted^7,9. Total number of root-galls/plant were counted in the infected roots of mulberry plants^12,15. The total protein content of the leaf and root samples (10 at random sampling / plot) from each of the 16 plots were determined^13,14,15.  All the data from experiments were counted for statistical analysis by t-test. In this field trial, sacrifices of mulberry plants were not done due to well reported pathological characters from our previous experiments ^{4to 7,9,10}.

Preparation of crude extract

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 crude ethanolic extract of A. nilagirica called CinaMT (Mother tincture or original solution)^7,9_.

Preparation of potentized drug

The crude ethanolic extract of A. nilagirica, (CinaMT) was diluted with 90% ethanol (1:100) in the proportion in a round vial. The vial was filled up to two-thirds of its space, tightly crocked and then was given 10 powerful downward strokes of the arm. This process of mechanical agitation is called succussion. 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 potency with 90% ethanol in the same proportion (1:100) and the given mixture was given 10 powerful down word strokes^7,9,16,17 .  In this way potencies up to Cina 200C and Cina 1000C are prepared.

Preparation of medicated Cina globules

Cina homeopathic potencies in liquid form can be kept in globules. A vial is filled up to two-third 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 crocked 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 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 and such medicated globules are known to retain their properties for many years^10,16,17 . In this process the drug soaked globules; Cina MT, Cina 200C and Cina 1000C were prepared. The control medicated globules were similarly prepared with sucrose globules soaked in 90% ethanol^10,17.

Preparation of 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. The control solution was similarly prepared^10,17.

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 the other three were treated as treatment set. To assess the direct effect of Cina- test solution, the water was removed by pipette and in all the treatment sets, immediately replaced by 1ml of test solutions: CinaMT, Cina 200C and Cina 1000C (7.2 mg globules/ml concentration) were added respectively. The control set received 1 ml of control solution and observed at 30 minute intervals for a period of 12 hours exposure period at room temperature (25±2°C)^9,18. This mortality  test was replicated five times.

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Subhas Chandra Datta

Dr. Subhas Chandra Datta is Headmaster & Researcher, Eco-Club Research Unit, Kanchannagar D.N.Das High School, Kanchannagar, Burdwan, West Bengal, India. He has 14 years teaching experience and has been publishing research for 18 years. He is an expert in the identification of diseases and the methodology of that research. Dr. Subhas Chandra Datta is also a member of numerous professional societies including the Society for Biological Chemists, the Zoological Society of Burdwan and Calcutta Univ., and the Social and Environmental Biological Association.