Agricultural and horticultural compositions inducing resistance in plants against salt- and water-stresses

Abstract: The present invention relates to an agricultural and horticultural composition inducing resistance in plants against salt- and water-stresses which comprises as an active ingredient an effective amount of at least one cyclic dipeptide of the following formula (I) or an agriculturally or horticulturally acceptable salt thereof; wherein each of R₁ and R₂, which may be the same or different, is a hydrogen atom or lower alkyl group, or R₁ and R₂ are joined to form pyrrolidine ring which may optionally have hydroxy group, and R₃ is a hydrogen atom or hydroxy group.

Description: The use of certain cyclic deipeptides for inducing resistance in plants against salt- and water-stresses. The present invention relates to the use of certain cyclic dipeptides for inducing resistance in plants against salt- and water-stresses and to a method of inducing such resistance, especially by treating seeds. Among various factors causing damage to plants, drought and salt damage in coastal regions can obstruct plant growth by subjecting the roots to abnormal osmotic pressures. In such environments, plants are subjected to salt- and water-stresses. There is a need for substances having a normalizing effect on germination and the growth of seedlings under such stresses to prevent decrease of agricultural yields and the injury of garden plants. The invention is based on the discovery that certain cyclic dipeptides are very effective in inducing resistance in plants against salt- and water-stresses. Central Patents Index Basic Abstracts Journal, abstract no. J5 90093-081-A and JP-A-84 093 081 (1984) describe cyclo(L-Hypo(OR)-L-Pro) and cyclo(D-allyp(OR)-D-Pro), where R can be H, C₁₋₅ alkyl or acyl or benzyl as being effective for accelerating germination and root growth. Ienaga et al. describe in Tetrahedron Letters, vol 28, no. 12, March 1987, pages 1285-1286 L-hydroxyprolyl-L-proline anhydride and L-hydroxyprolyl-L-leucine anhydride as promoting rice germination at 20°C under dark conditions. According to one aspect the invention provides the use of a cyclic dipeptide of the formula (I) or an agriculturally or horticulturally acceptable salt thereof for inducing resistance in plants against salt- and water-stresses wherein each of R₁ and R₂, which may be the same or different, is a hydrogen atom or a C₁ to C₅ alkyl group, or R₁ and R₂ are joined to form pyrrolidine ring which may optionally have a hydroxy group, and R₃ is a hydrogen atom or hydroxy group. According to another aspect, the invention provides a method of inducing resistance in plants against salt- and water-stresses which comprises treating seeds with a cyclic dipeptide of the formula (I) or an agriculturally or horticulturally acceptable salt thereof as specified above. The concentration of the cyclic dipeptide or agriculturally or horticulturally acceptable salt thereof is preferably from 1 x 10⁻³ to 1 x 10⁻¹¹ M. In the above formula (I), each of R₁ and R₂, which may be the same or different, is a hydrogen atom or a straight or branched alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl or tert-pentyl, or R₁ and R₂ are joined to form pyrrolidine ring which may optionally have hydroxy group, and R₃ is a hydrogen atom or hydroxy group. The compounds of the formula (I) in the present application are represented using the abbreviations of amino acids which are adopted by IUPAC and IUB or are commonly used in the art to which the present invention pertains. The following abbreviations of amino acids and substituent groups are used. Pro: proline Ala: alanine Leu: leucine Hyp: hydroxyproline Me: methyl group nBu: n-butyl group The amino acid residue in the present invention may be any of the D-isomer, L-isomer and DL-isomer. Also aHyp represents the trans-isomer of Hyp between the headbridge hydrogen and hydroxy group. For example, cyclo[D-(Me)Leu-L-Hyp] is the cyclic dipeptide represented by the following formula. Preferred cyclic dipeptides of the present invention include: Cyclo(L-Ala-L-Pro) Cyclo(L-Leu-L-Pro) Cyclo(L-Ala-L-Hyp) Cyclo(L-Ile-L-Hyp) Cyclo(L-Leu-L-Hyp) Cyclo[L-(N-Me)Leu-L-Hyp] Cyclo[D-(N-Me)Leu-L-Hyp] Cyclo[D-(N-nBu)Leu-L-Hyp] Cyclo(L-Pro-L-Hyp) Cyclo(D-Pro-D-aHyp) Cyclo(D-Pro-D-Hyp) Cyclo(L-Pro-L-aHyp) Cyclo(L-Hyp-L-Hyp Cyclo(L-aHyp-L-aHyp) ±Cyclo(Pro-Pro) The cyclic dipeptides of the present invention also include agriculturally or horticulturally acceptable salts of the compounds of the above formula (I), for example, salts with alkali metal such as sodium or potassium, with alkaline-earth metal such as calcium, magnesium or barium, or with other metals such as aluminum and the like; salts with an acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, trifluoroacetic acid, formic acid, acetic acid, citric acid or lactic acid; or salts with an organic base such as ammonia or organic amine. These salts can be produced from free cyclic dipeptides in the usual way or can be interchanged with each other. The above cyclic dipeptides are known compounds, e.g. the compounds disclosed in the prior literatures, and so the compounds according to the present invention can be produced by art-recognized methods as follows: (1) Both of the two amino acids, such as Pro, Hyp, Ala, Leu, Ile and their derivatives having a lower alkyl group, which constitute the cyclic dipeptides of the present invention are reacted in an appropriate solvent such as water or ethylene glycol at room temperature or, if desired, by heating to higher temperature to give the desired compounds of the invention. 2) In an appropriate solvent such as methylene chloride which does not inhibit the reaction, Pro or Hyp of which hydroxy group is protected by a conventional hydroxy-protecting group such as n-butyl are condensed with Pro or Hyp of which hydroxy group is protected, or with Ala, Leu or Ile having a conventional amino-protecting group such as benzyloxycarbonyl by the use of a condensing agent such as dicyclohexylcarbodiimide. After the condensation reaction, cyclization of the dipeptide can be carried out simultaneously with removal of protecting group, for example, catalytic reduction using palladium-carbon, to give the cyclic dipeptides of the present invention. (3) The compounds having a lower alkyl group as a substituent R₂ can be produced by a conventional alkylation, for example, the hydroxy group of Hyp is protected by a hydroxy-protecting group such as tetrahydrofuran, and then reacted with an alkylating agent such as alkylhalide to introduce a lower alkyl group into the R₂ position. The resulting compounds of the present invention can be purified by known methods such as distillation, chromatography and recrystallization. Identification is established through, inter alia , melting point, elemental analysis, IR, NMR, UV, mass spectrum, etc. The specific rotatory power was measured using a sodium lamp (λ=5893 Å). Examples According to the above-mentioned methods of the cyclic dipeptides, the following compounds of the present invention were prepared. [Compound 1] Cyclo(L-Ala-L-Pro) m.p.: 179 - 184 °C [α]²⁴: -142.5° (C=1, methanol) [Compound 2] Cyclo(L-Leu-L-Pro) m.p.: 164 - 167 °C [α]²⁴: -131.7° (C=1, methanol) [Compound 3] Cyclo(L-Ala-L-Hyp) m.p.: 205 - 210 °C [α]²⁵: -150.7° (C=1, methanol) [Compound 4] Cyclo(L-Ile-L-Hyp) m.p.: 161.5 - 162 °C [α]²⁵: -152.5° (C=1, water) [Compound 5] Cyclo(L-Leu-L-Hyp) m.p.: 178 - 179 °C [α]²⁵: -148.2° (C=1, water) [Compound 6] Cyclo(L-(N-Me)Leu-L-Hyp) m.p.: oily substance [α]²⁵: -88,7° (C=1.2, methanol) [Compound 7] Cyclo(D-(N-Me)Leu-L-Hyp) m.p.: oily substance [α]²⁵: +55.8° (C=1, methanol) [Compound 8] Cyclo(D-(N-nBu)Leu-L-Hyp) m.p.: 112 - 114 °C [α]²⁵: +88.0° (C=1.1, methanol) [Compound 9] Cyclo(L-Pro-L-Hyp) m.p.: 141 - 142 °C [α]²⁸: -134.4° (C=1, methanol) [Compound 10] Cyclo(D-Pro-D-aHyp) m.p.: 174 - 175 °C [α]²⁸: +90.8° (C=1, methanol) [Compound 11] Cyclo(D-Pro-D-Hyp) m.p.: 141 - 142 °C [α]²⁸: +134.0° (C=1, methanol) [Compound 12] Cyclo(L-Pro-L-aHyp) m.p.: 175 - 176 °C [α]²⁸: -90.4° (C=1, methanol) [Compound 13] Cyclo(L-Hyp-L-Hyp) [Compound 14] Cyclo(L-aHyp-L-aHyp) [Compound 15] ±Cyclo(Pro-Pro) The following descriptions serve to illustrative agricultural and horticultural studies of the compounds of the present invention. (I) Effects on rice seedlings under salt-stress caused by sodium chloride (NaCl) A filter paper soaked with an aqueous solution of a test agent was laid in a germination dish. Even-sized seeds of rice ( Oryza sativa L. japonica ) were selected, washed with an aqueous solution containing 0.5% Tween 20 and 1.0% sodium hypochlorite, and then dried. The gemination dishes were sowed with the said rice seeds. The seeds were treated with the aqueous solution of the test agent only during the germination period (4 days). The five seeds having equal root lengths were transplanted from germination dishes to water culture vessels with/without NaCl-solution, not containing any test drug. Lengths and weights of both shoots and roots were measured 7 days after transplantation. These tests (30°C, 24h daylight) were repeated 6 times, and the mean value and standard error (S.E.) were obtained. Shoot length (mm) Root length (mm) 0% NaCl 71.2 ± 1.60 57.3 ± 3.29 0.1% NaCl 78.0 ± 1.74 53.3 ± 2.61 0.2% NaCl 77.6 ± 1.63 39.7 ± 2.02 0.3% NaCl 79.2 ± 1.48 37.8 ± 1.48 0.5% NaCl 45.3 ± 1.82 33.8 ± 1.76 0.75% NaCl 34.7 ± 0.66 32.9 ± 1.07 1.0% NaCl 28.2 ± 0.47 34.0 ± 1.75 Table 1 shows that the rice growth are apparently inhibited by NaCl in the medium. The effects of the compounds of the present invention inducing resistance in rice against salt-stress are described below: Concentration of Compound 2 (M) Shoot length (mm) Root length (mm) 1.0% NaCl control 28.8 ± 0.39 36.1 ± 1.45 1 x 10⁻⁴ 30.4 ± 0.60 38.2 ± 1.60 1 x 10⁻¹⁰ 31.1 ± 0.60 37.3 ± 1.69 1.5% NaCl control 20.2 ± 0.56 35.2 ± 1.52 1 x 10⁻⁴ 22.3 ± 0.53 38.3 ± 1.89 1 x 10⁻¹⁰ 21.9 ± 0.53 39.2 ± 2.11 Concentration of Compound 4 (M) Shoot length (mm) Root length (mm) 1.0% NaCl control 20.7 ± 0.96 15.7 ± 0.85 5 x 10⁻⁶ 30.9 ± 1.24 19.9 ± 0.60 5 x 10⁻⁸ 27.1 ± 0.67 21.2 ± 0.68 1.5% NaCl control 12.3 ± 0.64 12.1 ± 0.42 5 x 10⁻⁸ 16.0 ± 0.75 14.9 ± 0.52 5 x 10⁻¹⁰ 16.3 ± 0.66 14.7 ± 0.52 Concentration of Compound 5 (M) Shoot length (mm) Root length (mm) 1.0% NaCl control 20.7 ± 0.96 15.7 ± 0.85 5 x 10⁻⁴ 28.7 ± 0.87 19.7 ± 0.69 5 x 10⁻⁸ 25.4 ± 0.83 20.5 ± 0.87 1.5% NaCl control 12.3 ± 0.64 12.1 ± 0.42 5 x 10⁻⁴ 17.1 ± 0.76 13.9 ± 0.61 5 x 10⁻⁸ 15.6 ± 0.28 15.7 ± 0.58 Concentration of Compound 9 (M) Shoot length (mm) Root length (mm) 1.0% NaCl control 20.7 ± 0.96 15.7 ± 0.85 5 x 10⁻⁴ 28.8 ± 0.99 19.5 ± 0.96 5 x 10⁻⁸ 27.1 ± 0.83 21.8 ± 0.86 1.5% NaCl control 12.3 ± 0.64 12.1 ± 0.42 5 x 10⁻⁶ 17.3 ± 0.42 15.9 ± 0.44 5 x 10⁻¹⁰ 16.0 ± 0.64 14.0 ± 0.49 Concentration of Compound 13 (M) Shoot length (mm) Root length (mm) 1.0% NaCl control 27.3 ± 0.55 29.2 ± 1.96 1 x 10⁻⁶ 29.4 ± 0.61 38.5 ± 1.66 1 x 10⁻⁸ 30.5 ± 0.59 38.1 ± 2.03 1.5% NaCl control 20.5 ± 0.40 31.3 ± 1.65 1 x 10⁻⁴ 21.4 ± 0.69 35.6 ± 2.28 1 x 10⁻⁸ 21.9 ± 0.40 37.9 ± 1.58 Concentration of Compound 14 (M) Shoot length (mm) Root length (mm) 1.0% NaCl control 27.3 ± 0.55 29.2 ± 1.96 1 x 10⁻⁶ 31.8 ± 0.57 43.2 ± 1.69 1 x 10⁻¹⁰ 30.4 ± 0.60 38.2 ± 1.57 1.5% NaCl control 20.5 ± 0.40 31.3 ± 1.65 1 x 10⁻⁴ 21.8 ± 0.58 35.0 ± 1.77 1 x 10⁻⁶ 22.6 ± 0.83 35.3 ± 1.93 Concentration of Compound 15 (M) Shoot length (mm) Root length (mm) 1.5% NaCl control 21.9 ± 0.50 39.0 ± 1.76 1 x 10⁻⁶ 24.2 ± 0.50 42.7 ± 1.65 (II) Effects on rice seedlings under water-stress caused by mannitol Using mannitol instead of NaCl in the similar manner (I) as mentioned above, the effects of the compounds of the present invention inducing resistance against water-stress were studied. An example of the results is shown in Table 9. (III) Effects on tomato seedlings under salt-stress caused by NaCl Tomato seeds were treated with the aqueous solution of the test agent during the germination period (4 days). 9 days after transplantation from germination dishes to water culture vessels, 0.5% NaCl-solution was added to the vessels. The tomato seedlings had been cultivated for 22 days in the culture medium containing NaCl. An example of the results is shown in Table 10. Concentration of Compound 9 (M) (25∼30°C, 12h daylight, n=12∼18) Dried stem and leaves weight (mg) 0% NaCl control 156.9 ± 13.4 0.5% NaCl control 80.2 ± 7.6 1 x 10⁻⁶ 111.4 ± 8.8 1 x 10⁻⁸ 141.4 ± 14.4 (IV) Effects on cucumber seedlings under salt-stress caused by NaCl Cucumber seeds were treated with the aqueous solution of the test agent during the germination period (3 days). 11 days after transplantation from germination dishes to water culture vessels, 0.5% NaCl-solution was added to the vessels. The cucumber seedlings had been for 25 days cultivated in the culture medium containing NaCl. An example of the results is shown in Table 11. Concentration of Compound 9 (M) (25∼32°C, 12h daylight, n=5) Dried stem weight (mg) 0% NaCl control 151.7 ± 11.36 0.5% NaCl control 81.9 ± 5.31 1 x 10⁻⁶ 104.9 ± 7.37 1 x 10⁻⁸ 95.5 ± 3.25 As shown by the above-mentioned results (Table 2 to 8), rice seeds treated with the compounds of the present invention added to the growth medium during the germination period had resistant effects against the growth inhibition by NaCl after the transplantation of rice seedlings from germination dishes to water culture vessels containing NaCl. Also the compounds of the present invention induced resistance in other plants such as tomato (Table 10) and cucumber (Table 11) against salt-stress. Particularly in the case of tomato, seedlings not treated with the compounds of the invention were weak and fell down, on the other hand the seedlings treated with the present compounds had firm stems and so did not fall down as well as the group cultivated in the medium without NaCl. Similarly the effect of the present compounds inducing resistance against the plant growth inhibition was observed when a sugar was used instead of NaCl (Table 9). Therefore, the seeds treated with the compounds of the present invention during the germination period have resistant effects against water-stress causing osmotically obstruction to the plant growth as well as against salt-stress. By the treatment of the seeds of agricultural plants beforehand with the compounds of the present invention, though the plants are exposed to a drought or salt damage, they can resist salt- or water-stresses. Therefore, agricultural and horticultural compositions comprising as an active ingredient an effect amount of at least one cyclic dipeptide of the present invention are very useful in regions where agricultural and horticultural plants fall into the state of salt- and/or water-stresses, for example, a salty region such as reclaimed land, coast land easily suffered from salt damage or a region closed by a desert susceptible to be damaged by a drought. The effects inducing resistance in plants against salt- and water-stresses of the agricultural and horticultural compositions of the present invention can be achieved by treating seeds with a solution of the present composition during only the germination period. The cyclic dipeptide solution may be of any convenient concentration, preferably the concentration of from 1 x 10⁻³ to 1 x 10⁻¹¹ M, to treat seeds to induce resistance in plants against salt- and water-stresses. An effective results can be obtained even at low concentration, for example the concentration of from 1 x 10⁻⁸ to 1 x 10⁻¹⁰ M, which is advantage economically.

Claims: The use of a cyclic dipeptide of the formula (I) or an agriculturally or horticulturally acceptable salt thereof for inducing resistance in plants against salt- and water-stresses wherein each of R₁ and R₂, which may be the same or different, is a hydrogen atom or a C₁ to C₅ alkyl group, or R₁ and R₂ are joined to form pyrrolidine ring which may optionally have a hydroxy group, and R₃ is a hydrogen atom or hydroxy group. Use according to claim 1 wherein R₃ is a hydrogen atom. Use according to claim 1 wherein R₃ is a hydroxy group. Use according to claim 1 wherein R₁ is a C₁ to C₅ alkyl group. Use according to claim 4 wherein R₂ is a hydrogen atom. Use according to claim 4 wherein R₂ is a C₁ to C₅ alkyl group. Use according to claim 1 wherein R₁ and R₂ are joined to form pyrrolidine ring. Use according to claim 7 wherein R₁ and R₂ are joined to form pyrrolidine ring having hydroxy group. Use according to claim 7 wherein said cyclic dipeptide is selected from cyclo(L-Ala-L-Pro), cyclo(L-Leu-L-Pro), cyclo(L-Ala-L-Hyp), cyclo(L-Ile-L-Hyp), cyclo(L-Leu-L-Hyp), cyclo[L-(N-Me)Leu-L-Hyp], cyclo[D-(N-Me)Leu-L-Hyp], cyclo[D-(N-nBu)Leu-L-Hyp], cyclo(L-Pro-L-Hyp), cyclo(D-Pro-D-aHyp), cyclo(D-Pro-D-Hyp), cyclo(L-Pro-L-aHyp), cyclo(L-Hyp-L-Hyp), cyclo(L-aHyp-L-aHyp), ±cyclo(Pro-Pro) and an agriculturally or horticulturally acceptable salt thereof. A method of inducing resistance in plants against salt- and water-stresses which comprises treating seeds with a cyclic dipeptide of the formula (I) or an agriculturally or horticulturally acceptable salt thereof as specified in any preceding claim. A method according to claim 10 wherein the concentration of the cyclic dipeptide or agriculturally or horticulturally acceptable salt thereof is from 1 x 10⁻³ to 1 x 10⁻¹¹ M.

Inventors: IENAGA KAZUHARU C O INSTITUTE, KUROHASHI MASAHARU C O INSTITU, NAKAMURA KO C O INSTITUTE OF B, IENAGA, KAZUHARU C/O INSTITUTE OF BIO-ACTIVE, KUROHASHI, MASAHARU C/O INSTITUTE OF BIO-ACTIVE, NAKAMURA, KO C/O INSTITUTE OF BIO-ACTIVE
Applicants: NIPPON ZOKI PHARMACEUTICAL CO, NIPPON ZOKI PHARMACEUTICAL CO. LTD.
Application year: 1989
Main classification: A01N43
IPC codes: A01N43, C07K5