ABIO Journal

Introduction

Mulberry is an important economical crop plants in sericulture and it grows under a wide range of ecological condition. It holds a special place as a major foreign exchange earner for many tropical and temperate countries. India secures the second position for the production of raw silk in the world, which is short about 30% to fulfill the home requirements [1]. Right from sprouting and throughout growing seasons, it is largely affected by a number of pathogens like plant parasitic nematodes, fungus, bacteria, virus and insects causing various diseases forming disease-complex and break the host resistance [1-19]. These pathogens are the main obstacles causing considerable loss in yield and nutritive value of mulberry foliage. Feeding of the diseased leaves affect the health of the silkworms adversely and the cocoon yield in terms of quality and quantity [5-7,17,18,20]. Root-knot disease, caused by Meloidogyne incognita (Kofoid & White) Chit wood, reduces 10-12% leaf yield in addition to affecting the leaf quality for silkworms feeding [1-18]. Leaf spots disease, caused by Cercosporam moricola (Cooke) fungus, losses 10-35% leaf yield reducing moisture, proteins adversely and ultimately the quality and quantity of cocoons. Phyllactinia corylea (Pers.) Karst fungus, causing powdery mildew disease, is the most common and wide spread economically important disease reducing 10-30% leaf yield and reducing the crude protein content by as much as 33%. The mosaic disease, caused by mosaic virus, are inward curling of leaves, particularly leaf margin and tip with chlorotic lesions on the leaf surface, stunted growth and suppressed leaf size [1-18]. Tukra disease, caused by Maconellicoccus hirsutus (Green) (Pseudococcidae), tremendously reduces the leaves have depleted in nutritive value and plant growth, leaf yield and leaf protein content significantly [1-18].

Recently, synthetic and chemical pesticides are the most effective means of control, but they are both expensive and environmentally unfriendly. For sustainability of agriculture therefore, farmers should divorce the synthetic and chemical pesticides strategy and marry the phytochemicals option which is non-toxic to man and the environment, biodegradable and affordable to the peasant farmer in the developing world [19,20]. The “Evils” of synthetic and chemical pesticides has been a major concern to environmentalists. Recently efforts have therefore been shifted towards the use of plant extracts against pathogens as alternative to synthetic compounds. But it is not cost effective and it affects our biodiversity conservation directly [21-25].

To overcome these situations, it has been already observed that the extract prepared from the funicles of Acacia auriculiformis A. Cunn and its pure compounds acaciasides (A&B), are effective in reducing mulberry diseases leaving no residual toxicity in the leaves to affect the growing silkworm larvae [1-3,17-19,25]. And recently it has also been observed that the use of Cina, prepared from the flowering meristems of Artemisia nilagirica (Clarke) pamp and Aakashmoni prepared from the funicles of Acacia auriculiformis A. Cunn. on mulberry reduced root-knot disease and enriched sericulture industry [10].

Aims and Objectives

The purpose of the present investigation is to confirm the efficacy of the homeopathic medicines; Cina 1000C and Aakashmoni 1000C are ameliorating root-knot disease of mulberry (Morus alba L., cv. S₁) caused by root-knot nematodes pathogens and also to find out if the Aakashmoni 1000C can reduce the four foliar diseases, caused by pathogens, under field condition. The foliar diseases were: leaf spot disease, fungus pathogens, powdery mildew disease, mosaic disease and tukra disease. The effects of the leaves of the Cina 1000C - and Aakashmoni 1000C - treated plants on the leaf consumption, growth of silk worm’s larvae, silk gland weight and Effective Rate of Rearing (ERR) were also observed.

In course of our experiments with anti-nematode agents, Aakashmoni 1000C, it was observed that the mulberry plants besides being infected with root-knot nematodes, were also naturally infected with above mentioned four foliar diseases (leaf spot, powdery mildew, mosaic viral and tukra disease). Thus, both the root-knot and foliar diseases, caused by various plant pathogens, were taken in to consideration during the evaluation of the effects of Aakashmoni 1000C. The result would be more realistic in terms of the potentiality of the Aakashmoni 1000C, use as potential bio-agents, in controlling various plant pathogens.

Materials and Methods

Site of the Experimental Plots

The field experiment was carried out at the Sriniketan Sericulture Composite Unit, Government of West Bengal, India where temperature was 28+5 ^°C and relative humidity was 75+5%. Soil and root samples [8-21,24,25] were taken at random from a sericulture field spreading over an area of 5.6 acre of land with a view to determining the extent and intensity of M. incognita nematode pathogen infestation. Later, three areas (in the same locality and climatic condition) each measuring 0.02 ha; one naturally root-knot disease infected- untreated field and other two naturally root-knot diseases infected Cina 1000C and Aakashmoni 1000C- treated field, were demarcated in the mulberry field where there were no soil differences as well as environmental factor.

The first area nematode infected (2863+55 J₂ / 1 kg of soil) sandy soil was mixed with yard manure (2:1 Vol/ Vol). Every day, at least 40 random sampling of moist Rhizospheric soil (200 g of soil i.e., each sample collected by making a hole of 1.8 cm wide and 6 cm deep) were done in the nematode infected area for 30 days and were assessed the M. incognita population [20,22] and this naturally infected soil-filled area, demarking untreated field, was replicated thrice.

The other two areas of naturally M. incognita infected sandy soil field was also 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 previous one. These naturally infected soil-filled areas, demarking treated fields, were also replicated thrice. Mature three years old mulberry cutting (average 25 cm length and 20 g fresh weight) collected from same sericulture field, were planted with a gap of 45 cm throughout the experimental fields where there were no soil difference 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 [19,21-23]. At least 80 number at random rhizospheric soil sampling (200 g in each sample) were collected from rhizospheric root-soil area of root (10-15 cm X 10-15 cm) and at least 40 number at random root sampling (2 g 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 [3,9,11,20,22].

After three months’ growth of mulberry, M. incognita population were estimated in the rhizospheric soil as well as roots [19-23] (at least 40 at random sampling in each area) of mulberry plants in each areas of mulberry field. The M. incognita infected mulberry plants were achieved growth of 50-60 cm in height. All the infected mulberry plants were divided in to batches. The batches were; untreated- batches, Cina 1000C - and Aakashmoni 1000C -treated batches and each batch have 8-plots (20 plants/plot).

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 135 days when their root-knot, leaf spot, powdery mildew, viral and tukra diseases were assessed [3-16,22,25]. The field trial was replicated three times.

Plant Pathogens Caused Mulberry Diseases

Root-Knot Disease

Rhizospheric soil and root sample were taken at random from all the infected plots. Meloidogyne incognita populations (10 samples / plot in each plant group) were estimated in the rhizospheric soil as well as roots [3-25] of infected mulberry plants. Total number and surface area of leaves of all plant groups. Total number of root-galls/plant were counted in the infected roots of mulberry plants [3-25]. The total protein content of the leaf and root samples (10 at random sampling / plot) from plots were determined [19,23]. All the data from experiments were counted for statistical analysis by student’s T-test. In this field trial, sacrifices of mulberry plants were not done due to well reported pathological characters from our previous experiments [3,17,18,21,22].

Foliar Diseases

The main foliar diseases, observed in the sericulture field, were: leaf spot disease, powdery mildew disease, mosaic disease and tukra disease. All the disease identified according to their characteristic symptoms by the experts concerned [1,3-25]. Diseased leaves of each type were counted in each plot [21]. The percentage of disease infection based on diseased leaf surface area [5,18,21].

Preparation of Homeopathic Mother Tincture (MT)

Air-dried and powdered flowering meristems of A. nilagirica and funicles of A. auriculiformis, 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 extracts were removed by evaporation at room temperature (25+2 ^°C). The residues were dried in a desiccator over anhydrous calcium chloride. The crude residues were dissolved in 90% ethanol at 1 mg/ml concentration and were formed homeopathic mother tincture of A. nilagirica called Cina MT and A. auriculiformis, named Aakashmoni MT (Original solution or crude extract) respectively [5-14,24]_.

Preparation of Potentized High Diluted Liquid medicine

The homeopathic mother tinctures of Cina MT and Aakashmoni MT were diluted respectively with 90% ethanol (1:100) proportionate in a round vial. The vial was filled up to two-third of its space, tightly corked. And then were given 10 powerful down ward strokes of the arm. This process of mechanical agitation is called succession. This was the 1^st centesimal potency named Cina 1C and Aakashmoni 1C. All the subsequent potencies were prepared by further diluting each potency with 90% ethanol in the same proportion (1:100) and the mixture were given 10 powerful down ward strokes. In this way potencies up to Cina 1000C and Aakashmoni 1000C were prepared respectively [5-14,24].

Preparation of Medicated Globules

Both the homeopathic potencies in liquid form can be kept in globules. A vial was filled up to two-third of its empty space with sucrose globules of a particular size. Few drops of a liquid potency of Aakashmoni 1000C-were poured in to the vial to just moisten all the globules. The vial was corcked and then shaken so that all globules were uniformly moistened. The cork was loosened, and the vial was 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 was turned upright, well corcked and kept in a cool dry place away from light. The dry globules were then being kept in a vial and medicated globules were known to retain their properties for many years. In this process the drug soaked globules Cina 1000C and Aakashmoni 1000C was prepared.

Preparation of Control Globules

A vial was filled up to two-third of its empty space with sucrose globules of a particular size. Few drops of 90% ethanol were poured in to the vial to just moisten all the globules. The vial was corked and then shaken so that all globules were uniformly moistened. The cork was 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 was 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. In this process the 90% ethanol soaked control sucrose globules were prepared. The control globules were prepared in the same way for comparison to the preparation of medicated Cina 1000C - and Aakashmoni 1000C - globules which were prepared with the 90% ethanol media [5-14,24].

Preparation of Test and Control Solutions

The drug soaked globules of Cina 1000C- and Aakashmoni 1000C- were then be mixed with sterile distilled water in the proportion of 7.2 mg globules/ml of water. 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 Cina 1000C- and Aakashmoni 1000C- control solutions were prepared for comparison to the preparation of test solutions [5-14,24].

Mortality Test

Three sets of cavity block with 1 ml distilled water containing 50 larvae (J₂) of M. incognita were taken; one set was treated as control and other two were treated as treatment sets of Cina 1000C- and Aakashmoni 1000C-. To assess the direct effect of Cina 1000C - and Aakashmoni 1000C- test solutions, the water was removed by pipette from all the treatment sets, and immediately replaced by 1ml of test solutions - Cina 1000C- and Aakashmoni 1000C- (7.2 mg globules/ml concentration) were added respectively. To assess the direct effect of control solution, the control set was 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 [3,9,19,20] was replicated five times. It was noted that both the control (without drugs) and treatment (with drugs) sets were received sucrose globules [9,10]. This mortality tests were replicated five times.

Treatment

Seventy-six days after pruning, of mulberry plants, all the treatment was done by foliar spray 10 ml/plant (7.2 mg/ml concentration) once daily for 15 days with Cina 1000C- and Aakashmoni 1000C- test solutions and control solutions 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. All Cina 1000C- and Aakashmoni 1000C- treated groups were received 10 ml/plant test solutions (7.2 mg Cina 1000C- and Aakashmoni 1000C - globules/ml concentration) respectively. The infected untreated with Cina- and Aakashmoni- (control) groups were similarly received 10 ml/plant control solutions (7.2 mg- 90% ethanol soaked globules/ml concentration) [2,6-14,20,22,25]. It is noted that the infected untreated with Cina- and Aakashmoni (controls), were not untreated, but treated with the solution made from sugar pills soaked in the alcohol medium. The infected untreated (controls) were only treated with the solutions made from sugar globules in the alcohol medium (i.e. without medicine Cina - and Aakashmoni). At fifteen days after the second treatment all the parameters of diseases were assessed again for each group. All the data were used for statistical analysis by student’s T-test.

Analysis of Residue

A Thin Layer Chromatography plate (TLC) was made with silica gel (34% by weight). Mulberry leaves, collected one day after 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 developer [5,24]. Here, the residues run in Thin Layer Chromatography plate (TLC) with the standard from the Cina 1000C- and Aakashmoni 1000C- test [5-12].

Rearing of Silkworms

The eggs of a mother moth of the multivoltine ‘Nistari’ race (Bombyx mori L.) supplied by Regional Sericultural Research and Training Institute, Berhampore-742101, India, after hatching (93% hatching rate) and brushing 1^st stage silk worm larvae in the rearing tray, the larvae were divided into three batches (180 silkworm larvae / batch) and reared [2,3-14,16]. The larvae of infected untreated batch (control) were fed with the leaves of pathogens infected diseased leaves of mulberry plants from infected untreated (control) plots and the larvae of infected treated two-batches were fed with the leaves of Cina 1000C- and Aakashmoni 1000C - treated leaves of mulberry plants from infected treated respectively. Fresh leaves were given to the larvae 4 times daily. Mulberry leaves were used for feeding fifteen days after the last treatment with both the drugs. 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 moulting, moulting span days and mortality rate were recorded. The fresh silk gland weight of mature 5^th instar larvae

(before start spinning), starting time to spinning, span of spinning, fresh cocoon weight, fresh shell weight, silk layer ratio (SR% = Shell weight / Cocoon weight X 100), Effective Rate of Rearing (ERR% = Number of cocoon harvested / Number of silk worm hatched X 100), sex ratio percentage (Number of male adult emerged / Number of female adult emerged X 100) and egg laying capacity of mother moth were determined [2,3-14,16]. For statistical analysis by student’s T-test, ten mature 5^th instars silkworm larvae for fresh silk gland weight and ten cocoons for fresh shell weight were dissected out in each batch including replica of all batches [1,3-14,21,24]. All the data from rearing trial were used for statistical analysis by student’s T-test.

Results

Estimation of the Nematode Population from Field Trial

The initial nematode populations, stretching over an area of 5.6 acre of mulberry plantation, were 1779±43 J₂ per 200g of soil and 830±45 J₂ per 2g of root. The nematode populations in the demarcated 0.16 acre, were 1950±11 J₂ per 200g of soil and 615±15 J₂ per 2g of root [before treatment (Day-0)].

Mortality Test

It was observed that high diluted Cina 1000C- and Aakashmoni 1000C- had no toxic effects on nematodes mortality within the exposure period of 12 hours at room temperature (25±2 ^°C). For this reason, no data were presented in the results section.

Analysis of Residue

There had left no toxic residues of Cina 1000C- and Aakashmoni 1000C- in all the infected -treated plants by Thin Layer Chromatography plate (TLC). For this reason, no data were presented in the results section.

Root-Knot Disease

Table 1 shows the effects of Cina 1000C- and Aakashmoni 1000C- on Meloidogyne incognita pathogens infected mulberry plants in a field trial replicated thrice (P<0.01 by ‘T’- test). All naturally infected plants (treated plant group) treated with Cina 1000C- and Aakashmoni 1000C - showed increase number and surface area of leaves, and higher protein content in leaves and root than infected untreated (control) plants (untreated plant group). In all infected Cina 1000C- and Aakashmoni 1000C- treated plants, the population of root-knot nematodes decreased significantly in rhizospheric soil and as well as in roots than infected untreated (control) plants. The number of root galls also decreased significantly after Cina 1000C- and Aakashmoni 1000C- treatment.

Foliar Diseases

Table 2 shows only the effects of Aakashmoni 200C on leaf spot, powdery mildew, mosaic viral and tukra diseases of mulberry plants in a field trial replicated thrice assessed initially (Day- 0) and after a period of 30 days (Day -30) by ‘T’- test (P<0.01). Aakashmoni 1000C significantly reduced the number of leaves infected with leaf spot, powdery mildew, mosaic viral and tukra as compared to the pre-treatment condition (Day- 0). The percentage of control achieved were 62.08 for leaf spot, 77.89 for powdery mildew, 64.91 for mosaic virus and 38.42 for tukra infection as compared to the pre-treatment level (Day- 0). In case of infected untreated plots leaf spot, powdery mildew, mosaic viral and tukra diseases showed naturally 27.80%, 17.76%, 29.37% and 21.20% reduction respectively, in 30 days (Day -30).

Effects on Feeding Silkworms

Table 3 shows the effects of Cina 1000C- and Aakashmoni 1000C- on diseased infected mulberry plants in a silkworm rearing and field trial replicated thrice on the feeding, growth and mortality of silkworms (P<0.01 by ‘T’-test). The average consumption of leaves by the 5^th instars, average number of feeding to cocoon formation, average number of feeding day to cocoon formation, average number of escaping- feeding during moulting and average moulting span days were less for Cina 1000C- and Aakashmoni 1000C- treated plants than for infected untreated (control) ones. The average mortality rate (%) was nil with Cina 1000C- and Aakashmoni 1000C- treated plants groups and 56% with infected untreated (control) one. However, the average fresh weight of the 5^th instars larvae were higher with Cina 1000C- and Aakashmoni 1000C- treated plants than with infected untreated (control) one.

Effects on Silk Production and Rearing Practices

Table 4 shows the effects of feeding Cina 1000C- and Aakashmoni 1000C- treated mulberry leaves on silk production, spinning characters and rearing practices in a silkworm rearing and field trial replicated thrice (P<0.01 by ‘T’-test). The average fresh silk gland weight, average fresh cocoon weight, average fresh shell weight and average Shell Ratio (SR%) were higher with Cina 1000C- and Aakashmoni 1000C- treated plants than with infected untreated (control) one. It is notable that average starting time to spinning day and average span of spinning day (i.e. duration of span) were fewer with the Cina 1000C- and Aakashmoni 1000C- treated plants than with infected untreated (control) ones. Average Effective Rate of Rearing (ERR%), average sex ratio percentage and average egg laying capacity were significantly higher with all Cina 1000C- and Aakashmoni 1000C- treated groups.

Discussion

The high diluted homeopathic drugs; Cina 1000C- and Aakashmoni 1000C- ones again confirm that the cost-effective drugs not only reduced root-knot, leaf spot, powdery mildew, viral and tukra diseases but also improved the nutritive value of the treated leaves of infected plants [10-16]. From this field trial, it is confirmed that Cina 1000C and Aakashmoni 1000C also improves the nutritive value of the treated leaves which directly influences on the consumption of leaves, number of feeding and number of feeding day to cocoon formation, and indirectly effects on moulting stage in all the Cina 1000C- and Aakashmoni 1000C -treated groups from these trials. And due to ill development of infected untreated (control) batches larvae took more time to moult which is proved from the number of escaping feeding during moulting [2,3,7-14].

Higher nutritive value of treated plants contributes to higher growth of silkworm larvae, silk gland weight, cocoon weight and shell weight which increase silk production significantly [2,3,7-14]. for commercial purpose. The improved health of the larvae, cocoon weight, silk gland and shell weight from the Cina 1000C- and Aakashmoni 1000C- treated groups of the infected plants might have resulted in the fewer starting time to spinning and span of spinning day and the total elimination of the mortality rate [2,3-14,24]. However, Cina 1000C- and Aakashmoni 1000C is too dilute to contain drug molecules [2,3,7-14,24]. Naturally, the drug might not have affected the nematode directly [3,7-14,24]. and for this reason, no mortality occurs.

The Effective Rate of Rearing (ERR%) is very high in all Cina 1000C- and Aakashmoni 1000C- treated treatment batches which enriches the sericulture industry in many ways, especially for commercial purpose [3,7-14,24]. The mulberry leaves did not contain any toxic residues of the Cina 1000C- and Aakashmoni 1000C- test substances by the Thin Layer Chromatography (TLC). It is reported that Cina and Aakashmoni at ultrahigh 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 diluents medium [2,6-10,36].

Rather, the drug Cina 1000C- and Aakashmoni 1000C- might have induced natural defense response in the test plants against nematode parasites and has conferred defense response on growing larvae [3,7-14,24]. In fact, it is surprising that all infected Cina 1000C- and Aakashmoni 1000C- treated plants not only are less affected by nematodes but also have a better growth than the infected untreated with Cina 1000C- and Aakashmoni 1000C (control) plants [3,7-14,25,24]. And the positive effects of growth may be responsible for defense resistance against pathogens. Both the drugs also improve the plant growth effectively which directly increase photosynthesis rate and significantly reduce CO₂ in the environment. So we can say that Cina 1000C- and Aakashmoni 1000C might have induced synthesis of many new proteins which have stimulated increased photosynthesis rate, stomatal activity and water retention capacity of Cina 1000C- and Aakashmoni 1000C- treated plants [2-18].

The positive effects of growth on infected Cina 1000C- and Aakashmoni 1000C- treated plants may not only be responsible for defense resistance to nematodes pathogen but also improves growth of silkworm larvae and silk gland weight, cocoon weight, shell weight and Effective Rate of Rearing (ERR%) [2-18]. which increase silk production for commercial purpose. It is proved from the result that silk production is higher in the Cina 1000C- and Aakashmoni 1000C- treated groups than infected untreated with Cina 1000C- and Aakashmoni 1000C- (control) groups [2-18].

Conclusion

These results once again suggest that plant diseases (like nematodes, fungus, virus, bacteria and insects etc.) might be effectively controlled by the potentized cost effective homeopathic medicines Cina 1000C- and Aakashmoni 1000C- at an extremely low dose and also increases silk production and effective rate of rearing commercially which directly enriches sericulture industry as well as agriculture sector. Both the potentized bio-pesticide homeopathy drugs also improve the plant growth effectively which directly increase photosynthesis rate and significantly reduce CO₂ in the environment. And these cost-effective homeopathic medicines easily available and biodegradable, non-phytotoxic and non-pollutant as well as conserve our biodiversity which will contain “Global Green, Growth and Green Economy I.e. Sustainable Environment, Health and Development”. Acknow

Acknowledgements

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 lastly, for help in statistical analysis we are immensely indebted to Dr. Tapan Mondal, Asst. teacher of Secondary School.

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