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 nilagiricaExtract (Cina); mulberry; root-knot; Silk; Effective rate of rearing; field trial.
Running head : “Potentized Artemisia nilagiricaExtract increase…..”.
Correspondence by conventional mail : Dr. Subhas Chandra Datta, Researcher & Assistant teacher., C/O- Rajendranath Nag, Bajeprotappur (Katwa Road), Burdwan-713101,West Bengal, India.
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Objectives : Here, we show the effects of Potentized Artemisia nilagirica Extract (Cina)soaked globuleson 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 Cinahad 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 nilagiricaExtract (Cina); mulberry; root-knot; Silk;Effective rate of rearing; field trial
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 nematodes1,2,3. 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 farmers4,5,6,7. 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 industry7,9. The crude 90% ethanolic extract of the flowering meristems of A. nilagirica (1mg/ml concentration) and its potency are marketed by homeopathic pharmacists as Cina7,9. Here, in this field trial and silkworm rearing, we confirm the effects of Cinasoaked globuleson 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 samples10,11,12,13 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 area3,11,12. 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 fields3,11,12,15. 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 soil11,12 as well as roots5,11,12,13 (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.
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 soil11,12 as well as roots5,11,12,13,15 of infected mulberry plants. Total number and surface area of leaves of all plant groups were counted7,9. Total number of root-galls/plant were counted in the infected roots of mulberry plants12,15. The total protein content of the leaf and root samples (10 at random sampling / plot) from each of the 16 plots were determined13,14,15. 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 1st 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 strokes7,9,16,17. In this way potencies up to Cina 200C and Cina 1000C are prepared.
Preparation of medicated Cinaglobules
Cinahomeopathic 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 years10,16,17. In this process the drug soaked globules; CinaMT, Cina 200C and Cina 1000C were prepared. The control medicated globules were similarly prepared with sucrose globules soaked in 90% ethanol10,17.
Preparation of test solutions
The drug soaked globules ofCinaMT,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 prepared10,17.
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.
Forty five days after pruning of mulberry plants, all the treatment 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 drenched with solutions. During spraying, the soil surface underneath each plant was covered with polyethylene sheet. All the CinaMT, Cina 200C and Cina 1000C treated groups received 10ml/plant test solutions (7.2 mg Cina globules/ml concentration) respectively and other healthy and infected- plant groups similarly received 10ml/plant control solutions (7.2mg- 90% ethanol soaked globules/ml concentration) respectively7,9,10. We told about healthy ( control) and infected ( control ). These controls were 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. All the data were used for statistical analysis.
Analysis of residue
Mulberry leaves, collected one day after the last treatment were homogenized in a blender and extracted with ethanol. The residue run in thin layer chromatography plate (TLC) with the standard from the Cina test substances. The test substances wereCinaMT,Cina200Cand Cina 1000C7,9.
Rearing of silkworms
The eggs of a mother moth of the multivoltine ‘Nistari‘ race(Bombyx mori L.), after hatching ( 91 % hatching rate ) and brushing 1st stage silk worm larvae in the rearing tray, the larvae were divided into 8 batches ( 80number / batch ) and reared 4,6,7,19. The larvae of batch 1 were fed with the leaves of healthy (control) plants, those of batch 2 with the leaves of healthy Cina MT treated plants, those of batch 3 with the leaves of healthy Cina 200C treated plants, those of batch 4 with the leaves of healthy Cina 1000C treated plants, those of batch 5 with the leaves of infected (control) plants, those of batch 6 with the leaves of infected CinaMT treated plants, those of batch 7 with the leaves of infected, Cina 200C treated plants and those of batch 8 with the leaves of infected Cina 1000C treated plants. 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 larvae were kept inside the rearing chamber at 27±2°C and 70 + 15% RH. 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 molting, molting span days and mortality rate were recorded. The fresh silk gland weight ( before start spinning ), starting time to spinning, span of spinning, 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 6,7,19. For statistical analysis by t- test, ten mature 5th instar larvae for fresh silk gland weight and ten cocoons for fresh shell weight were dissected out in each treatment batches including replica of all treatment batches6,7,19. All the data from rearing trials were used for statistical analysis ( t– test ).
Table1 shows the effects of Cina on healthy and Meloidogyne incognita infected mulberry plants in a field trial (P<0.01 by ‘t’– test). All healthy and infected plants treated with Cinashowed increased number and surface area of leaves, and higher protein content in leaves and root than infected (control) plants. It is also noted that number, surface area, leaf and root- protein content was significantly higher in all healthy Cinatreated plants than healthy (control) plants. In all infected Cinatreated plants, the population of root-knot nematodes decreased significantly more in rhizospheric soil as well as in roots than in infected (control) plants. The number of root galls also decreased significantly after Cinatreatment. It is observed that Cina had no direct toxic effects on nematodes mortality within the exposure period of 12 hours and it left no toxic residues of Cina in the treated plants7,9. It is interesting that treatment effects were the most pronounced with Cina 200C.
Table 2 shows the effects of Cinaon the healthy and M. incognita infected mulberry plants in a field trial on the feeding, growth and mortality of silkworms (P<0.05 by ‘t’-test). The average consumption of leaves by the 5th instars, average number of feeding to cocoon formation, average number of feeding day to cocoon formation, average number of escaping – feeding during molting and average molting span days were less for Cina treated plants than for healthy and infected (control) ones. The average mortality rate was nil with all Cina treated plants and 8% with healthy (control) and 30% with infected (control ) ones. However, the average fresh weight of the 5th instars larvae were higher with Cinatreated and healthy plants than with infected (control) one.
Table 3 shows the commercially increased silk production by effective treatment with Cina on the feeding of healthy and M. incognita infected mulberry leaves in a field trial (P<0.05 by ‘t’-test). The average fresh silk gland weight, average fresh cocoon weight, average fresh shell weight and average shell ratio ( Shell weight / Cocoon weight X 100) were higher with healthy and Cina– treated plants than with infected (control) one. It is interesting that the shell ratios were higher in healthy plants treated with Cina than with the infected Cina treated ones. It is also notable that average starting time to spinning day and average span of spinning day ( i.e. duration of span ) were fewer with the healthy and Cinatreated plants than with infected ( control ) ones. Average effective rate of rearing ( ERR%) were higher with all Cinatreated groups.
The homeopathic drug Cina once again confirms that the cost effective Cina not only reduced root-knot disease but also improved the nutritive value of the treated leaves of infected plants7,9,10. From this field trial, we once again confirm that Cinaalso improved the nutritive value of the healthy treated leaves7,9,10 which directly influences the consumption of leaves, number of feeding and number of feeding day to cocoon formation, and indirectly affects molting stage in the all Cina treated groups from this trial. And due to ill development of healthy and infected (control) batches larvae took more time to molt which is proved from the number of escaping feeding during molting. Higher nutritive value of treated plants contributed to higher growth of silkworm larvae, silk gland weight, cocoon weight and shell weight which increased silk production significantly7 for commercial purpose. The improved health of the larvae, cocoon weight, silk gland and shell weight from the Cinatreated groups of healthy and infected- plants might have resulted in the shortened starting time to spinning and span of spinning day and the total elimination of the mortality rate. However, Cina is too dilute to contain drug molecules10. Naturally, the drug might not have affected the nematode directly7,9,10 and for this reason, no mortality occurs. The effective rate of rearing ( ERR% ) is very high in all Cina treated treatment batches which enriches the sericulture industry in many ways, especially for commercial purpose. The mulberry leaves did not contain any toxic residues of the Cinatest substances by the thin layer chromatography (TLC). It is reported that Cina at ultra high dilution has physical basis in the form of charge transfer interaction and altered rate of tumbling in the specific part of the molecules of the diluent’s medium9,10,17. Rather, the drug Cinamight have induced natural defense response in the test plants against nematode parasites and has conferred defense response on growing larvae5 to 7,9,10,20.
In fact, it is surprising that all infected Cina treated plants not only are less affected by nematodes, but also have a better growth than healthy (control) plants. It is further confirmed by the Cina treated effects on healthy plants in order to confirm the more positive effect on growth than in healthy (control) groups7,9,10 . The positive effects of growth may be responsible for defense resistance against pathogens. Cina might have induced synthesis of many new proteins which have stimulated increase photosynthesis rate, stomatal activity and water retention capacity of healthy and Cinatreated plants. The positive effects of growth on infected Cinatreated plants might not only be responsible for defense resistance to the nematodes pathogen, but also improved growth of silkworm larvae and silk gland weight, cocoon weight, shell weight and effective rate of rearing (ERR%) which increase silk production for commercial purpose. It is proved from the result that silk production is higher in all the healthy Cina treated group than infected Cinatreated groups.
These results suggest that plant diseases might be effectively controlled by the potentized and cost effective homeopathic medicine Cina, at an extremely low dose. It commercially increases silk production which directly enriches the sericulture industry as well as the agriculture sector. Further, it is not only easily available, but also non-phytotoxic and non-pollutant and also conserves our biodiversity.
The work described here has been supported by Rtd. Prof. N.C.Sukul, Dept. of Zoology, Visva-Bharati and Joint Director, Sriniketan Sericultural Composite Unit, Sriniketan, Govt. of West Bengal and Mr. Achintya Mondal, Secretary, Unit of Oriental Association for Education and Research (BIMS, BMS,BIMLS & OIST),West Bengal, India. Lastly, for help in statically analysis we are immensely indebted to Mr.Ashis Mondal, Asst. teacher of Ajodhya High School.
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Dr. Subhas Chandra Datta, Researcher & Assistant teacher, C/O- Rajendranath Nag, Bajeprotappur (Katwa Road), Burdwan– 713101,West Bengal, India.