P-BOOSTER

Abstract

Disclosed are plant growth promotors of general formula (I).

##STR00001##

Claims

1. A method for promoting plant growth comprising utilizing a compound of the general formula (1) ##STR00020## with the following definitions: R.sup.6 hydrogen, C.sub.1-36-hydrocarbon residue which can contain one to three halogen atoms and/or one to seven heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, R.sup.1 hydrogen, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, it also being possible for R.sup.1 and R.sup.3 to be covalently linked to form a 5- to 8-membered heterocyclic ring, which can contain 1 or 2 halogen atoms and/or one to three further heteroatoms in addition to the heteroatoms N already part of the heterocyclic ring, with m 3;4; 5; 6 and at most two of R.sup.5 not being hydrogen R.sup.4 independently hydrogen, halogen, NO.sub.2, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur.

2. The method of claim 1, wherein the compound of the general formula (1) is a compound of the general formula (1a) ##STR00021## with the following definitions: R.sup.3 hydrogen, C.sub.1-30-hydrocarbon residue which can contain one to three halogen atoms and/or one to six heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, R.sup.1 hydrogen, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, it also being possible for R.sup.1 and R.sup.3 to be covalently linked to form a 5- to 8-membered heterocyclic ring, which can contain 1 or 2 halogen atoms and/or one to three further heteroatoms in addition to the heteroatoms N already part of the heterocyclic ring, ##STR00022## with m 3;4; 5; 6 and at most two of R.sup.5 not being hydrogen R.sup.4, R.sup.5 independently hydrogen, halogen, NO.sub.2, C.sub.1-12-hydrocarbon residue which can contain one or two halogen atoms and/or one to three heteroatoms, selected from the group consisting of nitrogen, oxygen and sulfur, n 0; 1; 2 or 3.

3. The method as claimed in claim 1 as a biostimulant and/or as a fertilizer for suppressing the expression of phosphate starvation biomarkers in plants or the SPX1 biomarker or the Arabidopsis gene SPX1.

4. The method as claimed in claim 1 for mitigating and/or reducing the phosphate starvation stress of plants or for improving the phosphorus or phosphate use efficiency of plants or the phosphorus or phosphate uptake of plants, the root hair development, root system architecture, drought resistance development, nutrient uptake efficiency of plants, the phosphate availability for plants, water or nutrient uptake of plants, beneficial symbiotic interactions of plants, soil microbiome or phosphorous or phosphate availability from phosphate rock or organic phosphate.

5. The method as claimed in claim 1, wherein in the compound of the general R.sup.1 is hydrogen or, preferably linear, C.sub.1-6-alkyl.

6. The method as claimed in claim 1, The method as claimed in claim 1, wherein in the compound of general formula (I) m is 3 or 4 and R.sup.5 is hydrogen.

7. The method as claimed in claim 1, wherein in the compound of the general formula (I) R.sup.3 is hydrogen or C.sub.1-6-alkyl.

8. The method as claimed in claim 1, wherein in the compound of the general formula (I) R.sup.3 contains one to three cyclic groups independently selected from 5- to 8-membered carbocyclic or heterocyclic groups that can be annelated.

9. A method for promoting growth of a plant, comprising adding to growing medium or soil in which the plant is grown, a compound as defined in claim 1.

10. A mixture containing at least one compound of general formula (I) as defined in claim 1, and at least one additional agrochemical agent, preferably selected from the group consisting of at least one inorganic and/or organic and/or organomineral fertilizer at least one nitrification inhibitor, preferably selected from the group consisting of 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, at least one urease inhibitor, preferably selected from N-n-butylthiophosphoric triamide (NBPT) and/or N-n-propylthiophosphoric triamide (NPPT), at least one customary agrochemical auxiliary agent, preferably selected from the group consisting of aqueous and/or organic solvents, pH-adjusting agents, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity-adjusting agents, emulsifiers, dispersants, sequestering agents, anti-settling agents, coalescing agents, rheology modifiers, defoaming agents, photo-protectors, anti-freeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anticaking agents, mineral and/or vegetable oils and/or waxes, colorants and drift control agents, and mixtures thereof.

11. A fertilizer mixture, containing A. an inorganic and/or organic and/or organomineral fertilizer and B. effective amount, preferably 10 to 10000 weight-ppm, based on the fertilizer, of a compound of the general formula (I) as defined in claim 1.

12. The fertilizer mixture as claimed in claim 10, wherein the fertilizer mixture is in solid form and the compound of the general formula (I) is incorporated into the fertilizer or is applied to the surface of the, preferably inorganic, fertilizer.

13. The fertilizer mixture as claimed in claim 10, wherein the fertilizer mixture contains at least one additional agrochemical agent, preferably selected from the group consisting of at least one nitrification inhibitor, preferably selected from the group consisting of 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methyl carbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, at least one urease inhibitor, preferably selected from N-n-butylthiophosphoric triamide (NBPT) and/or N-n-propylthiophosphoric triamide (NPPT), at least one customary agrochemical auxiliary agent, preferably selected from the group consisting of aqueous and/or organic solvents, pH-adjusting agents, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity-adjusting agents, emulsifiers, dispersants, sequestering agents, anti-settling agents, coalescing agents, rheology modifiers, defoaming agents, photo-protectors, anti-freeze agents, biostimulants, pesticides/plant production products, biocides, plant growth regulators, safeners, penetrants, anticaking agents, mineral and/or vegetable oils and/or waxes, colorants and drift control agents, and mixtures thereof.

14. A process for producing the fertilizer mixture as claimed in claim 11 by introducing the compound of the general formula (I) into the fertilizer, and/or applying the compound of the general formula (I) to the surface of the fertilizer.

15. A method of fertilizing soils exploited agriculturally or horticulturally, wherein a fertilizer mixture containing compounds A and B A. an inorganic and/or organic and/or organomineral fertilizer and B. 10 to 10000 weight-ppm, based on the fertilizer, of a compound of the general formula (I) as defined in claim 1, or compounds A and B separately, but within a period of 0 to 5 hours, preferably 0 to 1 hour, more preferably approximately at the same time, is applied to the soils.

Description

EXAMPLES

[0296] We report on a substance that suppresses Arabidopsis SPX1 and promotes plant growth in phosphate starvation conditions. We validate its positive effect on plant growth in diverse plants, and show that the activity of this substance is retained in a number of variants, which structurally characterize the class of molecules with this activity.

[0297] These results show that the use of SPX genes as a biomarker to screen for SPX suppressing molecules is a potent approach to discover new plant growth promoting or phosphate stress reducing substances.

I. Materials and Methods

1. Plant Material and Growth Conditions

[0298] All Arabidopsis plant experiments were performed using the Arabidopsis thaliana Columbia-0 background. The Arabidopsis pSPX1::GUS line is described in the Planet Journal 2008, 54: 965-975. For rice, the cultivar Nipponbare was used. For maize, the cultivars DKC 2931 and 8104 were used for regular pot experiments and sand-based pot experiments, respectively. For soybean, the cultivar Primus was used. Medium-based plant experiments used a modified Murashige and Skoog medium (modMS20 mM NH.sub.4NO.sub.3, 0.1 mM H.sub.3BO.sub.3, 3 mM CaCl.sub.2, 0.1 ?M CoCl.sub.2*6H.sub.2O, 0.1 ?M CuSO.sub.4*5H.sub.2O, 0.1 mM Na.sub.2EDTA*2H.sub.2O, 0.1 mM FeSO.sub.4*7H.sub.2O, 1.5 mM MgSO.sub.4*7H.sub.2O, 0.1 mM MnSO.sub.4*H.sub.2O, 1 ?M Na.sub.2MoO.sub.4*4H.sub.2O, 5 ?M KI, 19 mM KNO.sub.3, 0.03 mM ZnSO.sub.4*7H.sub.2O, 0.1 g/L myo-inositol and 0.5 g/L MES (pH 5,7)), or a regular half strength Murashige and Skoog medium (MS1/2) with 300 ?M phosphate (low phosphate: 300 ?M KH2PO4, 9.9 mM KCl), 600 ?M phosphate (suboptimal phosphate: 600 ?M KH.sub.2PO.sub.4, 9.6 mM KCl) or 1.25 mM phosphate (high phosphate: 1.25 mM KH.sub.2PO.sub.4), except when stated otherwise. The medium was solidified due to the addition of 0.6% Gelrite, unless stated otherwise. In case of vertical plates (root length and root hair measurements), the medium was supplied with 0.5% of sucrose and 1 ?M (low phosphate, 1 ?M KH.sub.2PO.sub.4, 9.9 mM KCl), 10 ?M (low phosphate 10 ?M KH.sub.2PO.sub.4, 9.9 mM KCl) or 1.25 mM KH.sub.2PO.sub.4 (high phosphate). Arabidopsis seeds were stratified for 2 days at 4? C. before germination. Seedlings were germinated in climate-controlled growth chambers at 22? C. under continuous light (100 N mol m.sup.?2 s.sup.?1). Husked rice seeds were germinated on wetted Whatman paper for 2 days at 37? C. and then transferred to Sterivent boxes or square Petri dishes with solid medium. Maize and soybean seeds were wetted 24 hours at room temperature prior to sowing in pots.

2. Chemical Activity Assessment

[0299] For the chemical activity assessment 3 to 8 seeds per well of Arabidopsis pSPX1::GUS line were sown in 96-well plates containing 150 ?l of low phosphate modMS liquid medium containing 1% of sucrose. Compounds were added in 50 ?M concentration, starting from a 5 mM stock of compounds solved in DMSO. Different chemical substances were separately added to different wells. Per plate, 16 control wells were used, 8 containing only the low phosphate medium and 1% DMSO, and 8 containing high phosphate medium and 1% DMSO. 7 days after germination, plates were scanned using a flatbed scanner (EPSON Expression 11000XL) and analyzed for leaf size. After scanning, the growth medium was replaced by GUS staining solution (see Plant Mol. Biol. Rep. 1994, 12: 37-42 for details) and incubated for 1.5 hours at 37? C. The plates were scanned a second time and analyzed for GUS signal area. Measurements of both leaf area and GUS signal area were conducted by filtering out and measure the green and blue signal, respectively using ImageJ software. If the green leaf area was 50% or less than the control, samples were omitted. Additionally, all results were visually screened for leaf size and GUS signal.

3. Growth Assays

[0300] Arabidopsis primary root growth experiments were done on MS1/2 on vertical standing plates and roots were measured 11 days after germination. Roots were imaged using a flatbed scanner (EPSON Expression 11000XL) and root length was measured using ImageJ software. Arabidopsis shoot biomass was determined in 11 days after germination and growth on vertical standing plates with modMS. Arabidopsis leaf area was measured 21 days after germination on horizontal plates with modMS medium. Plates were scanned from the top using a flatbed scanner (EPSON Expression 11000XL) and the visible leaf area was measured using ImageJ software. Rice plate experiments used vertically standing Petri dishes containing MS1/2 medium (low phosphate0.001 mM) solidified with 0.8% washed agar. Plant height was measured 11 days after germination. Rice box experiments used modMS medium and a low phosphate condition of 0.025 mM phosphate. Plant height was measured 13 days after germination. All growth experiments occurred with 10 ?M of compound and in low phosphate conditions, unless stated otherwise. Controls always received the same DMSO concentration.

4. Pot Experiments

[0301] Regular Arabidopsis, soybean and maize pot experiments used an unfertilized potting soil, which was supplemented at the start of the experiment with 50 ml H.sub.2O containing 0.25 g/l NH.sub.4NO.sub.3 and 0.44 g/l (high phosphate) or no (maize and soybean) or 0.11 g/l KH.sub.2PO (Arabidopsis) for low phosphate conditions. For the sand-based maize pot experiment, sand was supplemented with 50 ml H.sub.2O containing 0.25 g/l NH.sub.4NO.sub.3 and 0.38 g/l (high phosphate) or 0.02 g/l (low phosphate) KH.sub.2PO.sub.4. Potassium was balanced via addition of KCl. Compound concentration was 10 ?M, calculated based on the total water content after watering, and was added at the start of the experiment (Arabidopsis) or once a week (maize). Indexed chlorophyll contents were received via a SPAD 502 Plus Chlorophyll Meter. All parameters in the sand experiment, except the rate of leaf 4 (measured the first 4 days after emergence), were measured 19 days after sowing. Measurements in the pot experiments with potting soil were done after 4 weeks of growth, except for leaf 5, which was measured at 26 days. The long term maize pot experiment was performed on the automated Phenovision platform (see Mertens et al, 2021, https://doi.org/10.3389/fpls.2021.640914) with maize cultivars B104 and DKC2931. End point measurements occurred after 90 days (B104) or 70 days (DKC2931). Plants were grown on an unfertilized potting soil, which was fertilized bi-weekly with 50 mL modified Hoagland solution (2.5? concentrated; but with 0.25 mM KH.sub.2PO.sub.4).

5. Phosphate Quantification

[0302] Col-0 ecotype Arabidopsis seeds were sown in 96-well plates containing 150 ?l of low phosphate modMS liquid medium containing 1% of sucrose. Compounds was added in 50 ?M concentration (1% DMSO). 7 days after germination, plates were scanned using a flatbed scanner (EPSON Expression 11000XL) and wells containing non-germinated seedlings were excluded from the following analysis. Plants were removed from each well and 50 ?l of medium was recovered from each well and used for molybdenum blue staining as described in Plant Physiology 2008, 146:1673-1686. To measure the phosphate level in soil, Olsen P extraction was used on 1 g of air dried soil, 500 ul of extract was used for molybdenum blue staining as described in The Plant cell 2013, 25:1641-1656. Values were expressed relative to the low phosphate control.

6. Anthocyanin Extraction and Quantification

[0303] Col-0 ecotype Arabidopsis seeds were sown in 96-well plates containing 150 ?l of low phosphate modMS liquid medium containing 1% of sucrose. Compounds was added in 50 ?M concentration (1% DMSO). Eight plants per repeat were collected 7 days after germination and put into a fresh Eppendorf. Extraction and quantification of anthocyanin levels was carried out according to The Plant cell 2013, 25:1641-1656. Anthocyanin levels were quantified by measuring absorption at 530 and 657 nm using a spectrophotometer and normalized to fresh weight input, and expressed relative to the low phosphate control. For pot experiments, the same procedure was followed with one 4-week old plant per sample.

7. Root Hair Length Quantification

[0304] Roots were imaged using a stereomicroscope or a VHX-6000. Number and length of the root hairs parallel to the medium surface was determined at 4 to 6 mm above the root meristem. Root hair length was measured using the ImageJ software.

II. Results

1. Compound 17 Reduces Phosphate Starvation

[0305] To identify new substances that suppress the expression of the SPX1 phosphate starvation biomarker, we used an Arabidopsis SPX1 reporter line pSPX1::GUS. After 7 days of growth in low phosphate conditions, the SPX1 biomarker, visualized by blue GUS staining, is typically clearly induced in the roots. But the molecule (named compound 17, see example 1 in Table 2) practically completely suppressed the SPX1 biomarker, and hence reduces phosphate starvation response. This observation was confirmed in two experimental repeats.

2. The Application of Compound 17 and Example 2 Reduces Phosphate Starvation Stress

[0306] As compound 17 showed the strongest suppression of SPX1, we selected this compound to further validate its effect on the reduction of phosphate starvation.

[0307] Two typical plant responses towards phosphate deficiency are the production of anthocyanin and growth inhibition of the primary root. To confirm the reduction of the phosphate starvation effect by compound 17, its effect on primary root growth and anthocyanin levels were evaluated in Arabidopsis.

[0308] Upon treatment with 10 ?M compound 17, the primary root length of 11-day-old Arabidopsis plants grown in low phosphate is almost double the length of the control without compound (Table 1). Moreover, the anthocyanin level in the leaves is reduced after treatment with compound 17, both when growing the plants in pots or in a 96-well plate assay (Table 1). These observation shows that compound 17 reduces the phosphate starvation response.

TABLE-US-00001 TABLE 1 Parameters related to phosphate starvation stress or growth are affected by compound 17 and example 2. Control indicates low phosphate conditions without compound (but with compound solvent), unless stated otherwise. Treatment Com- Plant and pound 17/ growth Exam- Exam- system Parameter Control ple 1 ple 2 Arabidopsis Primary root length (cm) 1.8 3.4 NA vertical plate Shoot biomass (mg) 11 21 NA Root hair number in high 3 9 8 phosphate conditions (one plane in 2 mm region) Average root hair length 0.11 0.26 0.28 per root (mm) in high phosphate conditions Total root hair length per 0.08 0.26 0.27 root (cm) in high phosphate conditions Arabidopsis Leaf area (mm.sup.2) 78 94 NA horizontal plate Leaf area (mm.sup.2) in 104 120 NA suboptimal phosphate conditions Arabidopsis Rosette size (cm.sup.2) 17 17 NA pot Phosphate level in soil 1 0.8 NA (relative value) Relative anthocyanin level 1 0.7 NA Arabidopsis Relative anthocyanin level 1 0.6 NA 96-well plate Rice plate Plant height (cm) 14.4 15 NA Rice box Plant height (cm) 1.1 4.1 NA Maize sand Plant height (cm) 21 27 NA Growth rate of leaf 4 2.04 2.64 NA (cm/day) Indexed chlorophyll 28 31 NA content Crown root number 2 8 NA Maize pot Shoot weight (g) 4 4 4.5 Plant height (cm) 57 59 62 Leaf 5 (cm) 40 43 46 Maize pot Biomass (g) 28 NA 37 (long term - B104) Plant height (cm) 708 NA 719 Sum of leaf length (cm) 5431 NA 6050 Plants with developed 31 NA 62 ears (%) Maize pot Biomass (g) 27 NA 31 (long term - DKC2931) Plant height (cm) 941 NA 1006 Sum of leaf length (cm) 3595 NA 3949 Plants with developed 100 NA 100 ears (%) Soybean pot Plant height (cm) 50 63 NA Biomass (g) 4.1 4.4 NA NA: Not Analyzed

3. The Application of Compound 17 Etc. Leads to Improved Plant Growth

[0309] To further validate the positive effect of compound 17 etc., growth experiments were performed in different growing systems and using different plant species. In different experiments, compound 17 etc. showed a clear positive effect on the shoot biomass, leaf area or plant height in Arabidopsis, rice, soybean and maize, both in plate, box or pot experiments, and in maize, ear formation was stimulated (Table 1). The phosphate levels in the soil after 4 weeks of Arabidopsis growth, show a reduction if treated with compound 17 etc. (Table 1). This indicates that compound 17 etc. treatment results in higher phosphate uptake.

[0310] Interestingly, we also observed that compound 17 etc. increased both the number of root hairs and the average length of root hairs, which together result in an increased total root hair length (Table 1). Root hairs and the root hair surface are known to be important for phosphate uptake. The induction of the root hairs and the accompanying increase in absorptive area might therefore explain the positive and phosphate starvation reducing effect of compound 17 etc.

4. A Set of Compound 17-Related Molecules Retains Activity and Reduces Phosphate Starvation

[0311] To explore the chemical space of the new phosphate fertilizer enhancer, a variety of structural homologs of compound 17 were characterized by evaluating their effect on the SPX1 phosphate starvation biomarker and/or on root hair development (Table 2, 3). This shows that structures with the general formula (I) retain compound 17 activity and reduce phosphate starvation.

[0312] Finally, we identified a number of molecules that fits the formula (I) and effectively suppressed the SPX1 biomarker in the primary screen (Table 2). This gives numerous examples of molecules included in the claimed class that show phosphate starvation stress reducing activity. A subset of these molecules were also used in an Arabidopsis growth experiment using horizontal plates, which showed that they effectively positively affect shoot growth (Table 4).

TABLE-US-00002 TABLE 2 Compound 17 and structural variants and their effect on SPX1 suppression (as represented by the GUS stained area) and/or total root hair length. Average GUS (SPX1) stained area GUS (SPX1) normalized to the Total root stained area number of seeds hair length normalized of low phosphate per root (in % to the number of controls in the same compared to the Example Structure seeds (pixels) plate (pixels) control) 1 (compound 17) [00007] 0.3 1578 492 2 [00008] 21 515 1009 3 [00009] NA 222 4 [00010] NA 193 5 [00011] NA 208 6 [00012] 8 1494 746 7 [00013] NA 434 8 [00014] NA 343 9 [00015] NA 210 10 [00016] 298 2208 NA 11 [00017] 800 2377 NA 12 [00018] NA 842 13 [00019] NA 799 NA: data not available

TABLE-US-00003 TABLE 3 Total root hair length per root (in % compared to the control) for different concentrations of example 1, 2 and 9 1 ?M 3 ?M 10 ?M Example 1 749 2184 2108 Example 2 886 1035 1784 Example 9 390 313 758

TABLE-US-00004 TABLE 4 Average leaf area of Arabidopsis plants grown on horizontal plates with different variants of compound 17 Example Leaf area (mm.sup.2) Control (no compound) 63 1 (compound 17) 66 3 69 4 72 5 65