| DE3735365A1 | 1988-04-21 |
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CLAIMS
1. An aqueous composition to stimulate growth and defense against pathogens and other favorable productive properties in plants, characterized in that it comprises an oligo-carrageenan selected from kappal, kappa2, lambda or iota oligo-carrageenans .
2. The aqueous composition of claim 1, characterized in that it comprises 0.5, 1 or 5 mg/mL of an oligo-carrageenan selected from kappal, kappa2, lambda or iota oligo-carrageenans.
3. The aqueous composition of claim 2, characterized in that it comprises 0.5 mg/mL of an oligo-carrageenan selected from kappal or kappa2 oligo-carrageenans.
4. The aqueous composition of claim 2, characterized in that it comprises 5 mg/mL of an oligo-carrageenan selected from iota oligo-carrageenan.
5. The aqueous composition of claim 2, characterized in that it comprises 1 mg/mL of an oligo-carrageenan selected from lambda oligo-carrageenan.
6. A method to stimulate growth and defense against pathogens and other favorable productive properties in plants, characterized in that it comprises the application of the aqueous composition of any one of claims 1 to 5 by spraying plant leaves .
7. The method of claim 6, characterized in that it comprises spraying the aqueous composition of any one of claims 1 to 5 on the upper and lower parts of plant leaves.
8. The method of any of claims 6 or 7, characterized in that the plant is a tobacco plant.
9. The method of any of claims 6 to 8, characterized in that spr-aying is done once a week, at least three times, and growing of the plant continues .
10. The method of claim 9, characterized in that the plants are grown at least for the next 45 days after treatment.
11. The method of claim 10, characterized in that the plants are grown at ambient temperature.
12. The use of the composition of any one of claims 1 to 5, characterized in that it is useful to stimulate growth and defense against pathogens in plants .
13. The use of claim 12 characterized in that it is useful to stimulate the defense against TMV in plants.
14. The use of claim 12 characterized in that it is useful to stimulate defense against TMV in tobacco plants.
15. The use of claim 12 characterized in that it is useful to increase the height, the foliar biomass and the number of leaves in a plant.
16. The use of claim 15, characterized in that it is useful to increase the height, the foliar biomass and the number of leaves in a tobacco plant.
17. The use of claim 12, wherein the aqueous composition comprises 0.5 mg/mL of kappal or kappa2 oligo-carrageenans, characterized in that it is useful to increase the foliar biomass in a plant.
18. The use of claim 12, wherein the aqueous composition comprises 0.5 mg/mL kappal oligo-carrageenan, characterized in that it is useful for increasing the number of leaves in a plant.
19. The use of claim 12, wherein the aqueous composition comprises 5 mg/mL lambda oligo-carrageenan, characterized in that it is useful for increasing the number of leaves in a plant.
20. The use of claim 12 wherein the composition comprises 1 or 5 mg/mL lambda or iota oligo-carrageenans, characterized in that it is useful for increasing the defense against TMV infection in a plant.
21. The use of claim 22, characterized in that it is useful for increasing the defense against TMV infection in tobacco plants.
23. Preparation method of the aqueous composition of any one of claims 1 to 5, characterized in that it comprises:
- using carrageenans obtained from Chilean carrageenophyte seaweeds Sarcothalia crispata and Gigartina skotsbergll solubilizing the carrageenans in hot water depolymerizing the carrageenans by acid hydrolysis to obtain fractions of approximately 20 units of sulphated galactose in distinct positions of the galactose ring corresponding to kappal, kappa2, lambda and iota oligo-carrageenans .
24. The method of claim 23, characterized in that it further comprises performing the depolymerization of kappal, kappa2, iota and lambda oligo-carrageenans by hot acid hydrolysis adding hydrochloric acid to an aqueous solution of carrageenans at a concentration of 10 mg/mL, such as not to significantly alter the degree of sulphatation of the depolymerized products; and stop the depolymerization reaction by adding sodium hydroxide to obtain a pH equal to 7.0.
25. The method of claim 26, characterized in that it comprises concentrating the oligo-carrageenans using dialysis bags and sucrose outside to extract the water; drying the semisolid product to dryness in an oven at 40 0 C; pulverizing the solid product in a mortar; and solubilizing it in cold distilled water. by proxy ALEJANDRA LEONOR SOLANGE MOENNE MUNOZ |
COMPOSITION AND METHOD TO STIMUIATE GROWTH AND DEFENSE AGAINST PATHOGENS IN PLANTS
The present application relates to a composition and method to stimulate growth and defense against pathogens and other favorable productive properties in plants. Specifically, it relates to an aqueous composition of kappal, kappa2, lambda or iota oligo-carrageenans, to the treatment of plants, and to their properties to stimulate growth and defense against pathogens. These oligo-carrageenans are polymers of around 20 units of galactose linked by alternate α-1,4 and β-1,3 bonds with sulphatations on carbons 2 or 4 of the A ring (first galactose) and/or on positions 2 or 6 of the B ring (second galactose) . The aqueous composition of each of the four oligo-carrageenans is sprayed on plant leaves in order to stimulate growth, defense against pathogens and other productive properties.
Previous Art
For over five years, efforts have been made to prepare depolymerized fractions from polysaccharides extracted from Chilean marine algae showing favorable biological activities in plants, such as the stimulation of growth and the defense against pathogens. Initially, the effect of three fractions of oligosaccharides prepared via chemical hydrolysis from Chilean algae polysacharides were studied on the stimulation of growth and defense against tobacco mosaic virus (TMV) in tobacco plants (Laporte D., Vera, J., Chandia, N. P., Zύniga, E. A., Matsuhiro, B. y Moenne A., Structurally unrelated oligosaccharides
differentially stimulate growth and defense against tobacco mosaic virus in tobacco plants, Journal of Applied Phycology 19, 79-88, 2007) . The first fraction tested pertains to an oligosaccharide (PoIy-Gu), made of around 20 units of guluronic acid, prepared via acid hydrolysis of alginates obtained from the brown seaweed Lessonla trabeculata. The second fraction pertains to an oligosaccharide (PoIi-Ma) , made of around 20 units of mannuronic acid, prepared via acid hydrolysis of alginates obtained from the brown seaweed Lessonla vadosa. The third fraction pertains to an oligosaccharide (PoIi-Ga) , made of around 20 units of sulphated galactose with sulphatations on positions 2, 3 and 4 of the galactose ring, prepared via free radical hydrolysis of the sulphated galactan polymer obtained from the red seaweed Schyzlmenia binder!. Spraying of an aqueous solution of each of these oligosaccharides on tobacco leaves at a concentration of 0.5 mg/mL differentially stimulates growth and defense against TMV in tobacco plants (Xhanti NN) . Height and biomass of tobacco plants was particularly stimulated by PoIi-Ga and PoIi-Ma oligosaccharides, approximately 23% and 49% respectively, as compared with the controls sprayed with water. On the contrary, the PoIi-Gu showed no effect on growth. On the other hand, PoIi-Ga, PoIi-Ma and PoIi-Gu oligosaccharides stimulate defense against TMV infection by 74%, 22% and 9%, when analyzed 15 days after treatment. Therefore, PoIi-Ga oligosaccharide induces a slight stimulation of growth and a strong stimulation of defense against TMV in tobacco plants. These efforts gave rise to a patent filed on March 2005 at the
Chilean Industrial Property Office with Privilege Number 0693- 2005, currently under revision.
As PoIi-Ga oligosaccharide is difficult to obtain because the red seaweed Schyzimenia binder! is not abundant in the Chilean coast, the option of replacing PoIi-Ga by oligo-carrageenans obtained from commercial carragenans (Gelymar S. A.) was revised. These carrageenans are extracted from the red carrageenophytes seaweeds Sarcothalia crispata and Gigartina skotsbergii. Kappa 1, kappa 2, lambda and iota commercial carrageenans are galactose polymers attached by alternate bonds α-1,4 y β-1,3 with sulphatations on carbon 2 or 4 of the A ring (first galactose) and/or on position 2 or 6 of the B ring (second galactose) . The mentioned commercial carrageenans were solubilized in hot water and depolymerized by acid hydrolysis resulting in ' four fractions of around 20 units of sulphated galactose corresponding to the kappal, kappa2, lambda and iota carrageenans. These oligo-carrageenans were sprayed on the tobacco leaves at a concentration of 0.5, 1 and 5 mg/L and they induced a strong stimulation of growth and defense against TMV, when analyzed 45 days after treatment. Particularly, kappal and kappa2 oligo-carrageenans at a concentration of 0.5 mg/mL induced a significant increase of foliar biomass (between 133 and 175%) and lambda and iota oligo-carrageenans at a concentration of 1-5 mg/mL strongly protect tobacco plants (Xhanti NN) against TMV (80-95% protection) . It should be noted that the effect of each oligo-carrageenan is different from that of PoIi-Ga when assayed in tobacco plants, specially considering that all of them have a similar molecular weight, that the same concentration of
each was used, and that the effect was analyzed in the same cultivar of tobacco plants. This proves that oligo-carrageenans are agents that stimulate growth and defense against pathogens in plants different from PoIi-Ga. Moreover, although the four oligo- carrageenans are attached by α-1,4 y β-1,3 bonds and they differ only in sulphatation positions, their effect on growth and defense against pathogens in tobacco plants is also different, which proves that these oligo-carrageenans are essentially different agents from each other.
Brief description of the figures
Figure IA: Effect of distinct concentrations of kappal oligo- carrageenan on the height of tobacco plants (Xhanti NN) .
Figure IB: Effect of distinct concentrations of kappal oligo- carrageenan on the foliar biomass of tobacco plants (Xhanti NN) .
Figure 1C: Effect of distinct concentrations of kappal oligo- carrageenan on the number of necrotic lesions produced by tobacco mosaic virus infection in tobacco plants (Xhanti NN) .
Figure 2A: Effect of distinct concentrations of kappa2 oligo- carrageenan on the height of tobacco plants (Xhanti NN) .
Figure 2B: Effect of distinct concentrations of kappa2 oligo- carrageenan on the foliar biomass of tobacco plants (Xhanti NN) .
Figure 2C: Effect of distinct concentrations of kappa2 oligo- carrageenan on the number of necrotic lesions produced by tobacco mosaic virus infection in tobacco plants (Xhanti NN) .
Figure 3A: Effect of the distinct concentrations of iota oligo- carrageenan on the height of tobacco plants (Xhanti NN) .
Figure 3B: Effect of the distinct concentrations of iota oligo- carrageenan on the foliar biomass of tobacco plants (Xhanti NN) .
Figure 3C: Effect of distinct concentrations of iota oligo- carrageenan on the number of necrotic lesions produced by tobacco mosaic virus infection in tobacco plants (Xhanti NN) .
Figure 4A: Effect of distinct concentrations of lambda oligo- carrageenan on the height of tobacco plants (Xhanti NN) .
Figure 4B: Effect of distinct concentrations of lambda oligo- carrageenan on the biomass of tobacco plants (Xhanti NN) .
Figure 4C: Effect of distinct concentrations of lambda oligo- carrageenan on the number of necrotic lesions produced by tobacco mosaic virus infection in tobacco plants (Xhanti NN) .
Detailed description of the invention
Carrageenans kappal, kappa2, iota and lambda are polymers of sulphated galactose units linked by alternate α-1,4 and β-1,3 bonds with sulphatations in distinct positions of the galactose A and B rings (first and second galactose) . Kappal carrageenan has a sulphatation on carbon 4 of the A ring and an anhydrous bond between carbons 3 and 6 of the B ring. Kappa2 carrageenan has a sulphatation on carbon 4 of the A ring, a high percentage of sulphatation (40 to 60%) on carbon 2 of the B ring and an anhydrous bond between carbons 3 and 6 of the B ring. Iota carrageenan has a sulphatation on carbon 4 of the A ring, a sulphatation on position 2 of the B ring and an anhydrous bond between carbons 3 and 6 of the B ring. The lambda carrageenan has a high percentage of sulphatation on carbon 2 of A ring (70%) and
a sulphatation on carbon 2 and 6 of the B ring (this carrageenan has no anhydrous bond in the B ring) .
The depolymerization of kappal, kappa2, iota and lambda carrageenans was made by acid hydrolysis adding 4N hydrochloric acid to an aqueous solution of the carrageenan at a concentration of 10 mg/mL concentration hot—incubated. In order to avoid significant alteration of the degree of sulphatation in the depolymerized products, the time of hydrolysis was short. The depolymerization reaction was stopped by adding 4N sodium hydroxide until a pH equal to 7.0 was obtained. The depolymerization products (oligo-carrageenans) were analyzed by electrophoresis in a 2% agarose gel and stained with 1% Alciane Blue dye which binds to sulphated sugars. In every case, products of uniform sizes were obtained with an approximate weight of 8,500 Daltons (no family of different size products was obtained, as could have been expected) . This shows that the depolymerization products (oligo-carrageenans) pertain to oligosaccharides of approximately 20 units of sulphated galactose. The oligo-carrageenans were concentrated using dialysis bags and sucrose outside to extract water. Then, the semi-solid product was taken to dryness in an oven at 40 0 C. The solid product was pulverized using a mortar and easily solubilized in cold distilled water.
Oligo-carrageenans kappal, kappa2, iota and lambda were solubilized in water at a concentration of 0.5, 1 and 5 mg/mL and they were sprayed (1 mL) on the upper and lower side of leaves of
Xhanti NN tobacco plants (starting height being 3 cm) . A group of 10 tobacco plants (treatment group) was sprayed with an oligo- carrageenan at a certain concentration and a group of 10 plants was sprayed only with water (control group) . The sprayings were run once a week, three times in total, and then tobacco plants were grown for 45 days at ambient temperature (summer) . The height of each plant in the control group (n=10) and that of each plant in the treated groups (n=10) was determined as well as the number of leaves (n=10) and foliar biomass (n=5) . In addition, one leave of each plant (n=5) of the control group and of the treatment group was infected using abrasive carburundum and 100 μL of a tobacco mosaic virus (TMV) suspension at a protein concentration of 0.4 mg/mL. After five days, the number of necrotic lesions was counted in the control and treated groups (TMV infection produces necrotic lesions in Xhanti NN tobacco plants) and the percentage of decrease in the number of necrotic lesions (percentage of protection) was determined.
The results of these experiments showed that the four oligo- carrageenans stimulate the growth in height of tobacco plants, mainly kappal and kappa2 oligo-carrageenans at a concentration of 0.5 mg/mL, which induced an increase in height of 436% and 362%, respectively. Likewise, the four oligo-carrageenans increase the weight of foliar biomass, mainly kappal and kappa2 oligo- carrageenans at a concentration of 0.5 mg/mL and iota oligo- carrageenan at a concentration of 5 mg/mL, which increased the weight by 175%, 135% and 142%, respectively. Moreover, the four oligo-carrageenans increase the number of leaves, mainly kappal
oligo-carrageenan at a concentration of 0.5 mg/iriL and lambda oligo-carrageenan at a concentration of 5 mg/mL, which increased the number of leaves by 100% and 80 %, respectively. Finally, the four oligo-carrageenans induce protection against TMV infection, mainly lambda oligo-carrageenan at a concentration of 1 mg/mL and iota oligo-carrageenan at a concentration of 5 mg/mL, which increased protection by 96% and 80%, respectively. Therefore, the four oligo-carrageenans stimulate growth of tobacco plants, particularly kappal and kappa2 oligo-carrageenans, and defense against virus, particularly lambda and iota oligo-carrageenans.
The following tables show results obtained with kappal, kappa2, iota and lambda oligo-carrageenans used in stimulating growth and defense in Xhanti NN tobacco plants.
Table I. Height of Xhanti NN tobacco plants treated with distinct concentrations of kappal, kappa2, iota and lambda oligo-carrageenans and the percentage of difference in height.
Carrageenan κ2
Control 5.0 + 1.5 0
0.5 mg/mL 23.1 ± 2.6 362
1 mg/mL 18.6 + 2.3 272
5 mg/mL 17.4 ± 2.8 248
Carrageenan i
Control 5.0 + 1.5 0
0.5 mg/mL 10.2 + 2.5 104
1 mg/mL 14.8 + 1.5 196
5 mg/mL 19.0 ± 2.4 280
Carrageenan λ
Control 5.0 + 1.5 0
0.5 mg/mL 9.2 + 1.5 84
1 mg/mL 11.6 ± 2.3 132
5 mg/mL 20.0 + 1.2 300
Leaves of a group of 10 Xhanti NN tobacco plants (n=10) were sprayed with 1 mL of aqueous solution of kappa1, kappa2, iota and lambda oligo-carrageenans at a concentration of 0.5, 1 and 5 mg/mL and a control group of 10 plants (n=10)was sprayed only with water. The height (cm) of each plant in the control group and in the treated groups was determined and the average value and standard deviation were obtained in each case. The percentage of difference in height was calculated subtracting the average height of the control group from the average
height of each treated group and considering the height of the control group as 100%.
Table II. Foliar biomass of tobacco plants (Xhanti NN) treated with distinct concentrations of oligo-carrageenans and the percentage of difference in foliar biomass.
Leaves of a group of 10 Xhanti NN tobacco plants (n=10) were sprayed with 1 mL of aqueous solution of kappal, kappa2, iota and lambda oligo-carrageenans at a concentration of 0.5, 1 and 5 mg/mL and a control group of 10 plants (n=10) was sprayed only with water. The weight of leaves (g of fresh tissue) of each plant in the control group and in the treated groups was determined and the average value and the standard deviation were obtained in each case. The percentage of difference in foliar biomass was calculated by subtracting the average biomass of the control group from the average biomass of each treated group and considering the weight of the control group as 100%.
Table III. Number of leaves of Xhanti tobacco plants NN treated with different concentrations of oligo-carrageenans and the percentage of difference in the number of leaves.
Leaves of a group of 10 Xhanti NN tobacco plants leaves (n=10) were sprayed with 1 mL of aqueous solution of kappal, kappa2, iota and lambda oligo-carrageenans at a concentration of 0.5, 1 and 5 mg/mL and the control group of 10 plants (n=10) was sprayed only with water. The number of leaves of each plant in the control group and in the treated groups was determined and the average value and the standard deviation were calculated in
each case. The percentage of difference in the number of leaves was calculated by subtracting the average value in the control group from the average value of each treated group, and considering the number of leaves in the control group as 100%.
Table IV. Number of necrotic lesions in Xhanti NN tobacco plants treated with distinct concentrations of oligo-carrageenans and infected with tobacco mosaic virus and the percentage of protection against viral infection.
Leaves of a group of 10 Xhanti NN tobacco plants (n=10) were sprayed with 1 mL of aqueous solution of kappal, kappa2, iota and lambda oligo-carrageenans at a concentration of 0.5, 1 and 5 mg/mL and a control group of 10 plants (n=10) was sprayed only with water. The number of necrotic lesions in each plant of the control group and in the treated groups was determined and the average value and the standard deviation were calculated in each case. The percentage of protection against the TMV infection was calculated by subtracting the average value of necrotic lesions in each treated group from the average value of necrotic lesions in the control group and this difference turned into a percentage considering the control group as 100% infection or 0% protection.