Title of Invention	A ENZYME COMPOSITION FOR CONTROLLING INVESTATION OF WOOLY APHID AND SOOTY MOULD ON SUGARCANE PLANT AND A METHOD OF TREATMENT THEREOF
Abstract	The present invention deals with an enzyme composition usefUl for control of infestation of woolly aphid (Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants. Sugarcane woolly aphid has become the serious pest all over India. The woolly aphid multiplies profusely and desaps the foliage heavily and excretes honey dew like substances which favors the multiplication of sooty mould which results in drying of the leaves and ultimately loss in the yield and sugar recovery. Various commercial preparations have been used for the control of woolly aphid includes natural enemies and chemical pesticide. But all the above control measures are not found to be effective to control woolly aphid. The enzyme composition in the present invention as obtained from Myrothecium verrucaria, a deuteromycetes fungus, degrades both insect cuticle and fungal cell wall and is found to be effective in the control of woolly aphid infestation of sugarcane in laboratory as well as under field conditions and it also inhibits the growth of sooty mould.

Title of Invention

A ENZYME COMPOSITION FOR CONTROLLING INVESTATION OF WOOLY APHID AND SOOTY MOULD ON SUGARCANE PLANT AND A METHOD OF TREATMENT THEREOF

Abstract

The present invention deals with an enzyme composition usefUl for control of infestation of woolly aphid (Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants. Sugarcane woolly aphid has become the serious pest all over India. The woolly aphid multiplies profusely and desaps the foliage heavily and excretes honey dew like substances which favors the multiplication of sooty mould which results in drying of the leaves and ultimately loss in the yield and sugar recovery. Various commercial preparations have been used for the control of woolly aphid includes natural enemies and chemical pesticide. But all the above control measures are not found to be effective to control woolly aphid. The enzyme composition in the present invention as obtained from Myrothecium verrucaria, a deuteromycetes fungus, degrades both insect cuticle and fungal cell wall and is found to be effective in the control of woolly aphid infestation of sugarcane in laboratory as well as under field conditions and it also inhibits the growth of sooty mould.

Full Text	Field of the invention: The present invention relates to an enzyme composition useful for control of infestation of woolly aphid and sooty mould on sugarcane plants. More particularly the present invention relates to an enzyme composition useful for control of woolly aphid, Ceretovacuna lanigera, on sugarcane, or woolly apple aphid and mealy bug of grapes using enzyme mixture of Myrothecium verrucaria. The enzyme composition comprises mainly chitinases, lipases, proteases and (3-1,3 - glucanases. The present invention further relates to a method of preparation of the said enzyme composition useful for control of infestation of woolly aphid and sooty mould on sugarcane plants. The present invention also relates to a method for controlling infestation of woody aphid and sooty mould on sugarcane plants. Background and prior art of the invention; Sugarcane is a major industrial crop in India. The area under sugarcane in India and Maharashtra was about 42.97 and 5.95 lakh hectare respectively, during 2000 -2001 with average productivity of 69.6 and 84.4 tones per hectare and producing sugar 185.11 and 67.05 lakh tones, respectively (Vijay Kumar Sugar statistics Cooperative Sugar 2002, 34 (1), 57- 95). Maharashtra state alone contributes 35 % of total sugar production in India. The average production of sugarcane in Maharashtra has been declined drastically during last three years (Mahatma Phule Krishi Vidyapeeth, Rahuri, 2002). Now a day, sugarcane woolly aphid (Ceretovacuna lanigera) has become a serious pest in the state. The woolly aphid adults produce nymphs without mating (parthenogenesis). The woolly aphid multiplies profusely and desaps the foliage heavily and excretes honey dew like substances on the leaves which favors the multiplication of black sooty mould (Capnodium sp.), resulting in drying of leaves and ultimately decrease in yield and sugar recovery. These sugarcane leaves when used as a fodder for cattle resulted in cattle death due to the toxins produced by the sooty mould (Gupta and Goswamai Incidence of sugarcane woolly aphid and its effect on yield attributes and juice quality. Indian sugar, 1995, 44 (12), 883-885). The loss due to heavy incidence of woolly aphid was reported to be 26 % in sugarcane yield and 24 % in sugar content in Indonesia. (Farina, S. R. "Problems on WSCA and strategies for its control at Takakar sp."- Report to the manager of Takakar sp. 1994, Takakar sugar plantation, unpublished report, 9). Various commercial preparations have been used for the control of woolly aphid infestation on sugarcane such as Conobathra aphidivora Meyr. (Pyralidae:Lepidoptera), a potent predator that feeds on nymphs and adults of woolly aphid. However the multiplication rate of the predator is very low as compared to the woolly aphid infestation rate. Chemical control includes malathion and dimethoate sprays. These chemicals though effective in the control, woolly aphid reappear after 10-15 days and again multiply profusely on sugarcane leaves. The 0.1 % soap powder has also been tried to control woolly aphid, but it has scorching effect on sugarcane leaves. In case of biological control Verticillium lecanii was reported to control woolly aphid under field (personal communication). It controls 50% of woolly aphid in 15 days time. Till date, there was no report for use of enzyme mixture against woolly aphid on sugarcane. Myrothecium verrucaria, a deuteromycetes fungus produces extracellularly enzyme mixture, which degrades insect cuticle, when grown in a medium containing chitin as a sole carbon source within 7 days at 28° C under shaking condition. The enzyme mixture constitutes chitinase, lipase, protease and P-1, 3-glucanase Previously, this enzyme preparation was used to control a fungal plant pathogen Sclerotium rolfsii, a root infecting plant pathogenic fungus (Patil RS, Deshpande AM, Natu AA, Nahar P, Chitnis M, Ghormade V, Laxman R.S, Rokade S, Deshpande MV. Biocontrol of root infecting plant pathogenic fungus, Sclerotium rolfsii using mycolytic enzymes and chitin metabolism inhibitors singly and in combination. J. Bio. Control. 2001, 15, 157-164), and also reported for the control of mosquito larvae (Mendosa ES, Vartak PH, Rao JU, Deshpande MV. An enzyme from Myrothecium verrucaria that degrades insect cuticle for biocontrol of Aedes aegypti mosquito. Biotechnol Lett. 1996, 18, 373-376). The inventors of the present invention have highlighted the importance of application of enzyme mixture from M. verrucaria for the control of Helicoverpa armigera and Spodoptera litura, the lepidopteran polyphagous pests of pulses, cotton, vegetable crops, sugar beet etc. The enzyme complex plays an important role in the insect death due to the hydrolysis of insect cuticular chitin with the help of enzymes such as chitinase (S. Chavan, M. Kulye, M.Kulkarni and M.V. Deshpande. Myrothecium verrucaria: A potential biocontrol agent for the control of insect pest. Proceedings of National Symposium on Biomanagement of Insect Pests 2003, Annamalai nagar, India). In view of the above mentioned prior art, the inventors of the present invention realized that there exists a need for an enzyme composition which is highly effective in the control of woolly aphid and sooty mould on sugarcane plants. Objects of the invention; The main objective of the present invention is to provide an enzyme composition, which is effective in controlling the woolly aphid and sooty mould of sugarcane plants represented by general formula A(n)B(p)C(q)D(r); wherein A is a chitinase, B is a lipase, C is a protease and D is a p-1,3- glucanase.and n, p, q, r represent the concentrations of respective components. Another objective of the present invention is to provide a method of preparation of the said enzyme composition useful for control of infestation of woolly aphid and sooty mould on sugarcane plants. Yet another objective of the present invention is to provide a method for controlling infestation of woolly aphid and sooty mould on sugarcane plants. Summary of the invention: The present invention provides an enzyme composition useful for control of infestation of wooly aphid and sooty mould on sugarcane plants. The woolly aphid protective cover comprises of extensive woolly mass, especially containing wax (lipids) followed by the other insect cuticle components such as chitin and lipoproteins. It has been observed that the high levels of lipase activity in the enzyme composition plays a unique role in hydrolysis and separation of woolly mass from the insect body, thereby allowing the easy degradation of the insect cuticle with the help of other enzymes of the mixture in a short time. Accordingly, the present invention provides an enzyme composition useful for control of infestation of woolly aphid (Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants, wherein the said composition is represented by general formula (Formula Removed) wherein A is a chitinase, B is a lipase, C is a protease and D is a (3-1, 3- glucanase and n, p, q, r represent the concentrations of respective components. In an embodiment of the present invention, n is in the range of 5 - 10, p is in the range of 2-6, q is in the range of 0.2 -0.5 and r is in the range of 0.5 - 0.8, wherein n, p, q and r are expressed in terms of units of enzyme. In still another embodiment of the present invention, the ratio of A, B, C and D are in the range of 16:15:2:.05 to 20:17:2.5:0.1. In yet another embodiment of the present invention, the chitinase used is selected from the group comprising endochitinase, chitosanase, and W-acetyl (3-D-glucosaminidase in the range of 0.4 -0.6, 0.01-0.03 and 5-7 respectively, all expressed in terms of units of enzyme. In still another embodiment of the present invention, the said composition is prepared by mixing the commercially available components in the proportion given or optionally from culture supernatant of M verrucaria (MTCC 5191), wherein the said culture supernatant contains the said enzyme composition as claimed in claim 1. Further in another embodiment of the present invention, A method for controlling infestation of woolly aphid (Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants, comprising the following steps of: (a) culturing the Myothecium verrucarria (MTCC 5191) on medium containing chitin and fungal biomass at 24 -30°C for a period of 5-7 days at 140-180rpm; (b) filtering or centrifuging the culture obtained from step (a) at about 4000xg followed by the freeze dried to obtain culture supernatant: (c) incubating the sugarcane plant leaves infested with woolly aphids or sooty mould with the culture supernatant obtained from step (b) at about 25 degree C or about 4 days or optionally spraying enzyme composition: (d) calculating the percentage of mortality of wooly aphid and sooty mould to control the infestation of wooly aphid (Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants. In still another embodiment of the present invention, the microorganism used is Myothecium verrucarria having the strain designation MTCC 5191. In yet another embodiment of the present invention, the said medium comprising potato, dextrose and agar in the ration of 200:20:15. Further in another embodiment of the present invention, the chitin used is selected from the group on Indian chitin, sigma chitin, prawn shell waste, crab shell waste etc. In still another embodiment of the present invention, the culture supernatant contains chitinase, (3-1, 3-glucanase specific to swollen chitin, laminarin and casein, respectively as a substrate for estimating the enzyme activity. In yet another embodiment of the present invention, the culture supernatant contains lipase activity measured by Titrimetric assay. Brief description of the drawings; Figure 1 represents untreated woolly aphids of sugarcane. In figure 1 number 1 indicates the sugarcane leaf; number 2 indicates the infestation of woolly aphids on sugarcane leaf. Figure 2 represents the woolly aphids treated with enzyme composition of M verrucaria. In figure 2 number 1 indicates the sugarcane leaf while number 2 indicates the dead woolly aphids. Table 1 show the enzyme activities in M. verrucaria culture filtrate after 7 days. Table 2 shows the effect of different concentrations of CDE (Cuticle Degrading Enzymes) on the % mortality of woolly aphids of sugarcane under laboratory conditions. Table 3 shows the effect of enzymes of M. verrucaria on Capnodium sp. spore germination. Table 4 shows the effect of enzyme mixture of M verrucaria and Metasystoc, a chemical pesticide on woolly aphids of sugarcane under field conditions. Detailed description of the invention; The present invention provides an enzyme composition which is highly effective in the control of woolly aphid and sooty mould on sugarcane plants. The woolly aphid protective cover comprises of extensive woolly mass, especially containing wax (lipids) followed by the other insect cuticle components such as chitin and lipoproteins. It has been observed that the high levels of lipase activity in the enzyme composition plays a unique role in hydrolysis and separation of woolly mass from the insect body, thereby allowing the easy degradation of the insect cuticle with the help of other enzymes of the mixture in a short time. The present invention provides an enzyme composition useful for control of woolly aphid infestation on sugarcane plant, having a composition as represented by general formula given herein below (Formula Removed) wherein A is a chitinase, B is a lipase, C is protease, D is a P- 1,3- glucanase, n, p, q, and r represent the concentrations of respective components wherein (n) may be in the range of 5 to 10, (p) may be in the range of 2 to 6, (q) may be in the range, 0.2 to 0.5, (r) may be in the range 0.5 to 0.8 expressed in terms of units of enzyme. In one of the feature of the present investigation chitinase may be consisting of endochitinase, chitosanase and W-acetyl-p-D-glucosaminidase wherein the concentration of the above enzymes may be in the range of 0.4 to 0.6, 0.01 to 0.03, 5 to 7 expressed in terms of units of enzyme measured on acid swollen chitin, acid swollen chitosan and p- Nitrophenyl- W-acetyl-p-D-glucosaminide, respectively. The composition may be prepared by mixing the individual component commercially available in the proportion as stated herein before in the general formula. The composition may be alternatively obtained by growing aerobically M verrucaria in a conventional chitin medium for a period of 5 to 7 days at 24°C to 30°C under shaking conditions (140-180 rpm), harvesting the medium and separating the solid from the broth and obtaining the composition as solution in the separated liquid. Present invention also provides a method of treatment of sugarcane plants for controlling woolly aphid infestation which comprises spraying the aqueous solution of the composition or the solution of the composition obtained by the fermentation, on the sugarcane plants immediately on appearance of woolly aphids, repeating the spray within a period of 15 days, resulting in inhibiting and controlling the growth of woolly aphid infestation. The killing process of the aphid is as follows first the hydrolysis of woolly mass of aphid takes place due to the action of lipase of the enzyme composition then the hydrolysis of chitin (the main component of the cuticle) with the. help of chitinase of the enzyme composition. In feature of the present invention the spraying of aqueous solution of the composition or the solution obtained by the fermentation may be done by any conventional method of spraying of sugarcane plants using any conventional equipment for such spray. The following examples are given by the way of illustration of the present invention and should not construed to limit the scope of the present invention. Example 1 This example illustrates reproduction of composition by fermentation method Myrothecium verrucaria (MTCC 5191) was grown on the medium containing chitin such as Indian chitin, Sigma chitin, prawn shell waste, crab shell waste, fungal biomass (0.20%- 0.80% w/v) as a sole source of carbon at 28°C for 7 days at 140-180 rpm. The culture supernatant was collected after 7 days by filtration through cotton or by centrifugation at 4000xg. The culture filtrate thus obtained was then freeze dried. Chitinase, p-1, 3-glucanase and protease activities were estimated using acid swollen chitin (0.7% w/ v), laminarin (1% w/v) and casein (1% w/v), respectively as a substrate. Lipase activity was measured by titrimetric assay using olive oil emulsion as a substrate. Table 1: The enzyme activities in M. verrucaria culture filtrate after 7 days (Table Removed) Example 2 This example illustrates the efficiency of enzyme composition of M. verrucaria to control woolly aphid at different concentrations was monitored under laboratory conditions. The sugarcane leaves infested with woolly aphids were collected form the field. The leaf discs (3-5 cm) containing woolly aphids were kept in a petri plates containing moist filter paper. These woolly aphids were then sprayed with enzyme complex of M verrucaria using atomizer; the petri plates were incubated at 25°C for 4 days. The percent mortality was recorded daily up to 4 days. The Table 2 shows the % mortality of woolly aphid. The % mortality was calculated by using inactivated enzyme mixture, measured as lipase activity as a control. Table 2: Effect of different concentrations of CDE (Cuticle Degrading Enzymes) on the % mortality of woolly aphids of sugarcane under laboratory conditions (Table Removed) NM: No mortality Concentrations of CDE in the above experiment CDE 1: Lipase 1.0 U/ml, Chitinase 1.32 U/ml and Protease 0.08 U/ml CDE 2: Lipase 2.0 U/ml, Chitinase 2.51 U/ml and Protease 0.12 U/ml CDE 3: Lipase 3.0 U/ml, Chitinase 3.68 U/ml and Protease 0.17 U/ml CDE 4: Lipase 4.0 U/ml, Chitinase 4.96 U/ml and Protease 0.21 U/ml CDE 5: Lipase 5.0 U/ml, Chitinase 6.01U/ml and Protease 0.35 U/ml As the concentration of CDE increases % mortality of woolly aphid increases Example 3 This example illustrates the effect of enzyme complex on sooty mould (Capnodium sp). Capnodium spore suspension (107spores/ml) was prepared. To 1ml of the suspension 1ml of sterile distilled water (control) or 1ml of M verrucaria enzyme mixture (Chitinase 1.32 U/ml, Protease 0.08 U/ml, p-l,3-glucanase 0.207 U/ml and Lipase 1.0 U/ml) was added and kept at room temperature for 30min. These spore suspensions were then sprayed on YPG plates containing streptomycin (250mg/l). Plates were incubated at 28°C and observed time to time microscopically for % spore germination. Table 3: Effect of enzymes of M verrucaria on Capnodium sp. spore germination (Table Removed) Example 4 As a prerequisite the efficiency of enzyme composition of M verrucaria to control C. lanigera under field condition was monitored. The sugarcane leaves infested with white woolly aphids were sprayed with enzyme complex of M. verrucaria (Lipase 1.0 U/ml, Chitinase 1.57 U/ml, Protease 0.02 U/ml, ß-1,3-glucanase 0.207 U/ml) and the chemical treatment (Methyl Dimeton), was sprayed using power sprayer. The percent infestation was recorded daily up to 14 days. The Table 4 shows the % infestation of woolly aphids on the sugarcane leaves. Table 4: Effect of enzyme mixture of M. verrucaria and Metasystoc, a chemical pesticide on woolly aphids of sugarcane under field conditions (Table Removed) Number of sugarcane plants selected for observation: 3 Number of leaves per sugarcane plant: 3 Area of leaves for observation: Number of woolly aphids/ 2.5 cm x 2.5 cm on sugarcane leaf Advantages; The main advantages of the present invention are: 1) This enzyme mixture is effective in laboratory as well as under field conditions for the control of woolly aphid, Ceretovacuna lanigera of sugarcane. 2) This enzyme mixture controls the growth of sooty mould, Capnodium sp. 3) The enzyme mixture is biodegradable. 4) The enzyme mixture can be used singly or in combination (sequentially) with chemical 5) The reappearance of the pest is restricted. 6) It can dissolve the woolly mass and degrade the cuticle of aphids. We claim; 1. An enzyme composition useful for control of infestation of wooly aphid (Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants, wherein the said composition is represented by general formula (Formula Removed) wherein A is a chitinase, B is a lipase, C is a protease and D is a (3-1,3-glucanase and n, p, q, r represent the concentrations of respective components; wherein n is in the range of 5 - 10, p is in the range of 2-6, q is in the range of 0.2 -0.5 and r is in the range of 0.5 - 0.8, wherein n, p, q and r are expressed in terms of units of enzyme and wherein the ratio of A, B, C and D are in the range of 16:15:2:0.05 to 20:17:2.5:0.1; 2. An enzyme composition as claimed in claim 1, wherein the chitinase used is selected from the group comprising endochitinase, chitosanase, and N-acetyl p- D-glucosaminidase in the range of 0.4 -0.6, 0.01-0.03 and 5-7 respectively, all expressed in terms of units of enzyme. 3. An enzyme composition as claimed in claim 1, wherein the said composition is prepared by mixing the commercially available components in the proportion given in claim 1 or optionally obtained from culture supernatant of M verrucaria (MTCC 5191). 4. A method for controlling infestation of woolly aphid (Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants, wherein the said method comprising the following steps of: a. culturing the Myothecium verrucarria (MTCC 5191) on medium containing chitin and fungal biomass at 24 -30°C for a period of 5-7 days at 140-180rpm; b. filtering or centrifuging the culture obtained from step (a) at about 4000 x g followed by the freeze dried to obtain culture supernatant; c. incubating the sugarcane plant leaves infested with woolly aphids or sooty mould with the culture supernatant obtained from step (b) at about 25°C or about 4 days or optionally spraying enzyme composition; d. calculating the percentage of mortality of wooly aphid and sooty mould to control the infestation of wooly aphid (Ceretovacuna lanigera) and sooty mould (Capnoduim sp.) on sugarcane plants. 5. A method as claimed in claim 6, wherein the microorganism used is Myothecium verntcarria having the strain designation MTCC 5191. 6. A method as claimed in claim 4, wherein the said medium comprising potato, dextrose and agar in the ration of 200:20:15. 7. A method as claimed in claim 6, wherein the culture supernatant contains chitinase, p-1, 3-glucanase, and protease specific to swollen chitin, laminarin and casein respectively as substrate for estimating the enzyme activity and the lipase activity is measured by titrimetric assay. 8. An enzyme composition useful for control of infestation of woolly aphid (Ceretovacuna lanigera) and sooty mould (Capnoduim sp.,) on sugarcane plants and method thereof substantially as herein described with reference to the examples and figures accompanying this specification.

Full Text

Field of the invention:
The present invention relates to an enzyme composition useful for control of
infestation of woolly aphid and sooty mould on sugarcane plants.
More particularly the present invention relates to an enzyme composition useful for
control of woolly aphid, Ceretovacuna lanigera, on sugarcane, or woolly apple
aphid and mealy bug of grapes using enzyme mixture of Myrothecium verrucaria.
The enzyme composition comprises mainly chitinases, lipases, proteases and (3-1,3
- glucanases.
The present invention further relates to a method of preparation of the said enzyme
composition useful for control of infestation of woolly aphid and sooty mould on
sugarcane plants.
The present invention also relates to a method for controlling infestation of woody
aphid and sooty mould on sugarcane plants.
Background and prior art of the invention;
Sugarcane is a major industrial crop in India. The area under sugarcane in India and Maharashtra was about 42.97 and 5.95 lakh hectare respectively, during 2000 -2001 with average productivity of 69.6 and 84.4 tones per hectare and producing sugar 185.11 and 67.05 lakh tones, respectively (Vijay Kumar Sugar statistics Cooperative Sugar 2002, 34 (1), 57- 95). Maharashtra state alone contributes 35 % of total sugar production in India. The average production of sugarcane in Maharashtra has been declined drastically during last three years (Mahatma Phule Krishi Vidyapeeth, Rahuri, 2002). Now a day, sugarcane woolly aphid (Ceretovacuna lanigera) has become a serious pest in the state. The woolly aphid adults produce nymphs without mating (parthenogenesis). The woolly aphid multiplies profusely and desaps the foliage heavily and excretes honey dew like substances on the leaves which favors the multiplication of black sooty mould (Capnodium sp.), resulting in drying of leaves and ultimately decrease in yield and sugar recovery. These sugarcane leaves when used as a fodder for cattle resulted in cattle death due to the toxins produced by the sooty mould (Gupta and Goswamai Incidence of sugarcane woolly aphid and its effect on yield attributes and juice quality. Indian sugar, 1995,
44 (12), 883-885). The loss due to heavy incidence of woolly aphid was reported to be 26 % in sugarcane yield and 24 % in sugar content in Indonesia. (Farina, S. R. "Problems on WSCA and strategies for its control at Takakar sp."- Report to the manager of Takakar sp. 1994, Takakar sugar plantation, unpublished report, 9). Various commercial preparations have been used for the control of woolly aphid infestation on sugarcane such as Conobathra aphidivora Meyr. (Pyralidae:Lepidoptera), a potent predator that feeds on nymphs and adults of woolly aphid. However the multiplication rate of the predator is very low as compared to the woolly aphid infestation rate. Chemical control includes malathion and dimethoate sprays. These chemicals though effective in the control, woolly aphid reappear after 10-15 days and again multiply profusely on sugarcane leaves. The 0.1 % soap powder has also been tried to control woolly aphid, but it has scorching effect on sugarcane leaves. In case of biological control Verticillium lecanii was reported to control woolly aphid under field (personal communication). It controls 50% of woolly aphid in 15 days time.
Till date, there was no report for use of enzyme mixture against woolly aphid on sugarcane. Myrothecium verrucaria, a deuteromycetes fungus produces extracellularly enzyme mixture, which degrades insect cuticle, when grown in a medium containing chitin as a sole carbon source within 7 days at 28° C under shaking condition. The enzyme mixture constitutes chitinase, lipase, protease and P-1, 3-glucanase Previously, this enzyme preparation was used to control a fungal plant pathogen Sclerotium rolfsii, a root infecting plant pathogenic fungus (Patil RS, Deshpande AM, Natu AA, Nahar P, Chitnis M, Ghormade V, Laxman R.S, Rokade S, Deshpande MV. Biocontrol of root infecting plant pathogenic fungus, Sclerotium rolfsii using mycolytic enzymes and chitin metabolism inhibitors singly and in combination. J. Bio. Control. 2001, 15, 157-164), and also reported for the control of mosquito larvae (Mendosa ES, Vartak PH, Rao JU, Deshpande MV. An enzyme from Myrothecium verrucaria that degrades insect cuticle for biocontrol of Aedes aegypti mosquito. Biotechnol Lett. 1996, 18, 373-376).
The inventors of the present invention have highlighted the importance of application of enzyme mixture from M. verrucaria for the control of Helicoverpa
armigera and Spodoptera litura, the lepidopteran polyphagous pests of pulses, cotton, vegetable crops, sugar beet etc. The enzyme complex plays an important role in the insect death due to the hydrolysis of insect cuticular chitin with the help of enzymes such as chitinase (S. Chavan, M. Kulye, M.Kulkarni and M.V. Deshpande. Myrothecium verrucaria: A potential biocontrol agent for the control of insect pest. Proceedings of National Symposium on Biomanagement of Insect Pests 2003, Annamalai nagar, India).
In view of the above mentioned prior art, the inventors of the present invention realized that there exists a need for an enzyme composition which is highly effective in the control of woolly aphid and sooty mould on sugarcane plants.
Objects of the invention;
The main objective of the present invention is to provide an enzyme composition,
which is effective in controlling the woolly aphid and sooty mould of sugarcane
plants represented by general formula A(n)B(p)C(q)D(r); wherein A is a chitinase, B is
a lipase, C is a protease and D is a p-1,3- glucanase.and n, p, q, r represent the
concentrations of respective components.
Another objective of the present invention is to provide a method of preparation of
the said enzyme composition useful for control of infestation of woolly aphid and
sooty mould on sugarcane plants.
Yet another objective of the present invention is to provide a method for controlling
infestation of woolly aphid and sooty mould on sugarcane plants.
Summary of the invention:
The present invention provides an enzyme composition useful for control of infestation of wooly aphid and sooty mould on sugarcane plants. The woolly aphid protective cover comprises of extensive woolly mass, especially containing wax (lipids) followed by the other insect cuticle components such as chitin and lipoproteins. It has been observed that the high levels of lipase activity in the
enzyme composition plays a unique role in hydrolysis and separation of woolly mass from the insect body, thereby allowing the easy degradation of the insect cuticle with the help of other enzymes of the mixture in a short time.
Accordingly, the present invention provides an enzyme composition useful for control of infestation of woolly aphid (Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants, wherein the said composition is represented by general formula
(Formula Removed)
wherein A is a chitinase, B is a lipase, C is a protease and D is a (3-1, 3- glucanase and n, p, q, r represent the concentrations of respective components.
In an embodiment of the present invention, n is in the range of 5 - 10, p is in the range of 2-6, q is in the range of 0.2 -0.5 and r is in the range of 0.5 - 0.8, wherein n, p, q and r are expressed in terms of units of enzyme.
In still another embodiment of the present invention, the ratio of A, B, C and D are in the range of 16:15:2:.05 to 20:17:2.5:0.1.
In yet another embodiment of the present invention, the chitinase used is selected from the group comprising endochitinase, chitosanase, and W-acetyl (3-D-glucosaminidase in the range of 0.4 -0.6, 0.01-0.03 and 5-7 respectively, all expressed in terms of units of enzyme.
In still another embodiment of the present invention, the said composition is prepared by mixing the commercially available components in the proportion given or optionally from culture supernatant of M verrucaria (MTCC 5191), wherein the said culture supernatant contains the said enzyme composition as claimed in claim 1.
Further in another embodiment of the present invention, A method for controlling infestation of woolly aphid (Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants, comprising the following steps of:
(a) culturing the Myothecium verrucarria (MTCC 5191) on medium
containing chitin and fungal biomass at 24 -30°C for a period of
5-7 days at 140-180rpm;
(b) filtering or centrifuging the culture obtained from step (a) at
about 4000xg followed by the freeze dried to obtain culture
supernatant:
(c) incubating the sugarcane plant leaves infested with woolly
aphids or sooty mould with the culture supernatant obtained from
step (b) at about 25 degree C or about 4 days or optionally
spraying enzyme composition:
(d) calculating the percentage of mortality of wooly aphid and sooty
mould to control the infestation of wooly aphid (Ceretovacuna
lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants.
In still another embodiment of the present invention, the microorganism used is Myothecium verrucarria having the strain designation MTCC 5191.
In yet another embodiment of the present invention, the said medium comprising potato, dextrose and agar in the ration of 200:20:15.
Further in another embodiment of the present invention, the chitin used is selected from the group on Indian chitin, sigma chitin, prawn shell waste, crab shell waste etc.
In still another embodiment of the present invention, the culture supernatant contains chitinase, (3-1, 3-glucanase specific to swollen chitin, laminarin and casein, respectively as a substrate for estimating the enzyme activity.
In yet another embodiment of the present invention, the culture supernatant contains lipase activity measured by Titrimetric assay.
Brief description of the drawings;
Figure 1 represents untreated woolly aphids of sugarcane. In figure 1 number 1 indicates the sugarcane leaf; number 2 indicates the infestation of woolly aphids on sugarcane leaf.
Figure 2 represents the woolly aphids treated with enzyme composition of M verrucaria. In figure 2 number 1 indicates the sugarcane leaf while number 2 indicates the dead woolly aphids.
Table 1 show the enzyme activities in M. verrucaria culture filtrate after 7 days.
Table 2 shows the effect of different concentrations of CDE (Cuticle Degrading Enzymes) on the % mortality of woolly aphids of sugarcane under laboratory conditions.
Table 3 shows the effect of enzymes of M. verrucaria on Capnodium sp. spore germination.
Table 4 shows the effect of enzyme mixture of M verrucaria and Metasystoc, a chemical pesticide on woolly aphids of sugarcane under field conditions.
Detailed description of the invention;
The present invention provides an enzyme composition which is highly effective in the control of woolly aphid and sooty mould on sugarcane plants.
The woolly aphid protective cover comprises of extensive woolly mass, especially containing wax (lipids) followed by the other insect cuticle components such as
chitin and lipoproteins. It has been observed that the high levels of lipase activity in the enzyme composition plays a unique role in hydrolysis and separation of woolly mass from the insect body, thereby allowing the easy degradation of the insect cuticle with the help of other enzymes of the mixture in a short time.
The present invention provides an enzyme composition useful for control of woolly aphid infestation on sugarcane plant, having a composition as represented by general formula given herein below
(Formula Removed)
wherein A is a chitinase, B is a lipase, C is protease, D is a P- 1,3- glucanase, n, p, q, and r represent the concentrations of respective components wherein (n) may be in the range of 5 to 10, (p) may be in the range of 2 to 6, (q) may be in the range, 0.2 to 0.5, (r) may be in the range 0.5 to 0.8 expressed in terms of units of enzyme.
In one of the feature of the present investigation chitinase may be consisting of endochitinase, chitosanase and W-acetyl-p-D-glucosaminidase wherein the concentration of the above enzymes may be in the range of 0.4 to 0.6, 0.01 to 0.03, 5 to 7 expressed in terms of units of enzyme measured on acid swollen chitin, acid swollen chitosan and p- Nitrophenyl- W-acetyl-p-D-glucosaminide, respectively.
The composition may be prepared by mixing the individual component commercially available in the proportion as stated herein before in the general formula.
The composition may be alternatively obtained by growing aerobically M verrucaria in a conventional chitin medium for a period of 5 to 7 days at 24°C to 30°C under shaking conditions (140-180 rpm), harvesting the medium and separating the solid from the broth and obtaining the composition as solution in the separated liquid.
Present invention also provides a method of treatment of sugarcane plants for controlling woolly aphid infestation which comprises spraying the aqueous solution
of the composition or the solution of the composition obtained by the fermentation, on the sugarcane plants immediately on appearance of woolly aphids, repeating the spray within a period of 15 days, resulting in inhibiting and controlling the growth of woolly aphid infestation.
The killing process of the aphid is as follows first the hydrolysis of woolly mass of aphid takes place due to the action of lipase of the enzyme composition then the hydrolysis of chitin (the main component of the cuticle) with the. help of chitinase of the enzyme composition.
In feature of the present invention the spraying of aqueous solution of the composition or the solution obtained by the fermentation may be done by any conventional method of spraying of sugarcane plants using any conventional equipment for such spray.
The following examples are given by the way of illustration of the present invention and should not construed to limit the scope of the present invention.
Example 1
This example illustrates reproduction of composition by fermentation method Myrothecium verrucaria (MTCC 5191) was grown on the medium containing chitin such as Indian chitin, Sigma chitin, prawn shell waste, crab shell waste, fungal biomass (0.20%- 0.80% w/v) as a sole source of carbon at 28°C for 7 days at 140-180 rpm. The culture supernatant was collected after 7 days by filtration through cotton or by centrifugation at 4000xg. The culture filtrate thus obtained was then freeze dried. Chitinase, p-1, 3-glucanase and protease activities were estimated using acid swollen chitin (0.7% w/ v), laminarin (1% w/v) and casein (1% w/v), respectively as a substrate. Lipase activity was measured by titrimetric assay using olive oil emulsion as a substrate.
Table 1: The enzyme activities in M. verrucaria culture filtrate after 7 days
(Table Removed)
Example 2
This example illustrates the efficiency of enzyme composition of M. verrucaria to control woolly aphid at different concentrations was monitored under laboratory conditions. The sugarcane leaves infested with woolly aphids were collected form the field. The leaf discs (3-5 cm) containing woolly aphids were kept in a petri plates containing moist filter paper. These woolly aphids were then sprayed with enzyme complex of M verrucaria using atomizer; the petri plates were incubated at 25°C for 4 days. The percent mortality was recorded daily up to 4 days. The Table 2 shows the % mortality of woolly aphid. The % mortality was calculated by using inactivated enzyme mixture, measured as lipase activity as a control.
Table 2: Effect of different concentrations of CDE (Cuticle Degrading Enzymes) on the % mortality of woolly aphids of sugarcane under laboratory conditions

(Table Removed)
NM: No mortality
Concentrations of CDE in the above experiment
CDE 1: Lipase 1.0 U/ml, Chitinase 1.32 U/ml and Protease 0.08 U/ml
CDE 2: Lipase 2.0 U/ml, Chitinase 2.51 U/ml and Protease 0.12 U/ml CDE 3: Lipase 3.0 U/ml, Chitinase 3.68 U/ml and Protease 0.17 U/ml CDE 4: Lipase 4.0 U/ml, Chitinase 4.96 U/ml and Protease 0.21 U/ml CDE 5: Lipase 5.0 U/ml, Chitinase 6.01U/ml and Protease 0.35 U/ml As the concentration of CDE increases % mortality of woolly aphid increases
Example 3
This example illustrates the effect of enzyme complex on sooty mould (Capnodium sp). Capnodium spore suspension (107spores/ml) was prepared. To 1ml of the suspension 1ml of sterile distilled water (control) or 1ml of M verrucaria enzyme mixture (Chitinase 1.32 U/ml, Protease 0.08 U/ml, p-l,3-glucanase 0.207 U/ml and Lipase 1.0 U/ml) was added and kept at room temperature for 30min. These spore suspensions were then sprayed on YPG plates containing streptomycin (250mg/l). Plates were incubated at 28°C and observed time to time microscopically for % spore germination.
Table 3: Effect of enzymes of M verrucaria on Capnodium sp. spore germination
(Table Removed)
Example 4
As a prerequisite the efficiency of enzyme composition of M verrucaria to control C. lanigera under field condition was monitored. The sugarcane leaves infested with white woolly aphids were sprayed with enzyme complex of M. verrucaria
(Lipase 1.0 U/ml, Chitinase 1.57 U/ml, Protease 0.02 U/ml, ß-1,3-glucanase 0.207 U/ml) and the chemical treatment (Methyl Dimeton), was sprayed using power sprayer. The percent infestation was recorded daily up to 14 days. The Table 4 shows the % infestation of woolly aphids on the sugarcane leaves.
Table 4: Effect of enzyme mixture of M. verrucaria and Metasystoc, a chemical pesticide on woolly aphids of sugarcane under field conditions
(Table Removed)
Number of sugarcane plants selected for observation: 3
Number of leaves per sugarcane plant: 3
Area of leaves for observation: Number of woolly aphids/ 2.5 cm x 2.5 cm on
sugarcane leaf
Advantages;
The main advantages of the present invention are:
1) This enzyme mixture is effective in laboratory as well as under field
conditions for the control of woolly aphid, Ceretovacuna lanigera of
sugarcane.
2) This enzyme mixture controls the growth of sooty mould, Capnodium sp.
3) The enzyme mixture is biodegradable.
4) The enzyme mixture can be used singly or in combination (sequentially)
with chemical
5) The reappearance of the pest is restricted.
6) It can dissolve the woolly mass and degrade the cuticle of aphids.

We claim;
1. An enzyme composition useful for control of infestation of wooly aphid
(Ceretovacuna lanigerd) and sooty mould (Capnodium sp.) on sugarcane plants,
wherein the said composition is represented by general formula
(Formula Removed)
wherein A is a chitinase, B is a lipase, C is a protease and D is a (3-1,3-glucanase and n, p, q, r represent the concentrations of respective components; wherein n is in the range of 5 - 10, p is in the range of 2-6, q is in the range of 0.2 -0.5 and r is in the range of 0.5 - 0.8, wherein n, p, q and r are expressed in terms of units of enzyme and wherein the ratio of A, B, C and D are in the range of 16:15:2:0.05 to 20:17:2.5:0.1;
2. An enzyme composition as claimed in claim 1, wherein the chitinase used is
selected from the group comprising endochitinase, chitosanase, and N-acetyl p-
D-glucosaminidase in the range of 0.4 -0.6, 0.01-0.03 and 5-7 respectively, all
expressed in terms of units of enzyme.
3. An enzyme composition as claimed in claim 1, wherein the said composition is
prepared by mixing the commercially available components in the proportion
given in claim 1 or optionally obtained from culture supernatant of M
verrucaria (MTCC 5191).
4. A method for controlling infestation of woolly aphid (Ceretovacuna lanigerd)
and sooty mould (Capnodium sp.) on sugarcane plants, wherein the said method
comprising the following steps of:
a. culturing the Myothecium verrucarria (MTCC 5191) on medium
containing chitin and fungal biomass at 24 -30°C for a period of 5-7
days at 140-180rpm;
b. filtering or centrifuging the culture obtained from step (a) at about
4000 x g followed by the freeze dried to obtain culture supernatant;
c. incubating the sugarcane plant leaves infested with woolly aphids or
sooty mould with the culture supernatant obtained from step (b) at
about 25°C or about 4 days or optionally spraying enzyme
composition;
d. calculating the percentage of mortality of wooly aphid and sooty mould to control the infestation of wooly aphid (Ceretovacuna lanigera) and sooty mould (Capnoduim sp.) on sugarcane plants.
5. A method as claimed in claim 6, wherein the microorganism used is
Myothecium verntcarria having the strain designation MTCC 5191.
6. A method as claimed in claim 4, wherein the said medium comprising potato,
dextrose and agar in the ration of 200:20:15.
7. A method as claimed in claim 6, wherein the culture supernatant contains
chitinase, p-1, 3-glucanase, and protease specific to swollen chitin, laminarin
and casein respectively as substrate for estimating the enzyme activity and the
lipase activity is measured by titrimetric assay.
8. An enzyme composition useful for control of infestation of woolly aphid
(Ceretovacuna lanigera) and sooty mould (Capnoduim sp.,) on sugarcane plants
and method thereof substantially as herein described with reference to the
examples and figures accompanying this specification.

Documents:

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Patent Number

269370

Indian Patent Application Number

2378/DEL/2007

PG Journal Number

43/2015

Publication Date

23-Oct-2015

Grant Date

18-Oct-2015

Date of Filing

13-Nov-2007

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001,INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	MUKUND VINAYAK DESHPANDE	NATIONAL CHEMICAL LABORATORY, MAHARASHTRA.
2	SANTOSH BHIKULAL CHAVAN	NATIONAL CHEMICAL LABORATORY, MAHARASHTRA
3	MEDHA PRASHANTKULKARNI	NATIONAL CHEMICAL LABORATORY, MAHARASHTRA

PCT International Classification Number

C12P19/00; C12N9/24

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA