WO2015193781A1

WO2015193781A1 - Powdered fertilizer for supplementing boron deficiency in plants, fertilizing composition and use thereof

Info

Publication number: WO2015193781A1
Application number: PCT/IB2015/054484
Authority: WO
Inventors: José Felipe AQUEA ZEBALLOS; Jorge Patricio Arce Johnson
Original assignee: Pontificia Universidad Católica De Chile
Priority date: 2014-06-17
Filing date: 2015-06-12
Publication date: 2015-12-23

Abstract

The present invention relates to a boron-based fertilizer, specifically with a powdered fertilizer comprising methylboronic acid, macronutrients and micronutrients. This powdered fertilizer is useful for supplementing boron deficiency increasing leave and root growth, is highly soluble, so makes easy handling and application thereof, is not toxic to plants when applied at high concentrations and is innocuous to animal cells. In addition, a fertilizer composition and its use are described.

Description

POWDERED FERTILIZER FOR SUPPLEMENTING BORON DEFICIENCY IN PLANTS, FERTILIZING COMPOSITION AND USE THEREOF

INVENTION FIELD

The present invention relates to a boron fertilizer. In particular, the present invention relates to a powdered fertilizer comprising methylboronic acid as active ingredient for supplementing boron deficiency in plants, a composition of said powdered fertilizer and use of the same.

STATE OF THE ART

The role of boron element in plants was established as essential in 1923 by the publication of Warington, K. (1923), "The effect of boric acid and borax on the broad bean and Certain other plants" Annals of Botany 37, Number 629- 672. Boron is a structural component of cell wall and its absence results in zero plant growth as boron is essential in all processes of plant growth: buds, meristems, and roots. Besides, flowering and fruit development are very sensitive to boron availability. This implies that boron deficiency significantly affect crops, fruits and forest species yield, as described by Camacho-Cristobal, JJ, Rexach, J. and Gonzalez-Fontes, A. (2008) Boron in plants: deficiency and toxicity. J Integr Plant Biol, 50, 1247-125. Given the multiple functions of boron in plants, this element has been considered as one of the main plant micronutrients and its deficiency also occurs in many types of soils through the world.

One of the fundamental characteristics of boron deficiency in agriculture is that its lack inhibits plant growth tissues, especially reproductive structures representing 80% of world agricultural products. Fertilizing with boron is a common agricultural and silvicultural practice, especially in soils deficient in this micronutrient. Moreover, boron fertilizers tend to accumulate in drought conditions, becoming toxic to plants due to its excess in the soil. Boron deficiency is transient in many crops and therefore is difficult to prevent. In many areas of the world frequent treatments to prevent its deficiency have been incorporated, but these applications can become toxic due to environmental conditions and narrow range of concentration at which boron acts. There are several products on the market that are used for boron fertilization. This micronutrient are uptaken by plants from soil in the chemical form of boric acid. In some crops it is applied directly on the leaves and flowers, in fact, since the mid-1930s boric acid was the first product recommended for foliar applications.

Currently, boron main sources used as raw material in agriculture are: Borax (Na2B₄U7- 10H₂O), sodium octoborate (Na, Solubor) (Na2B₄O7+Na₂B ₀Oi6), boric acid (H₃BO₃), boron frits (Na₂B₄), boron ethanolamine (liquid), colemanite (Ca₂B₆Oii), ammonium pentaborate (NH₄B₅Os), ulexite (NaCaB₅O6(OH)₆) in the form of powder or liquid boron.

A high dose of soluble boron can produce toxic effects on plants, especially if boron-containing fertilizer is applied very close or highly concentrated, affecting growth and yield of plants. All products mentioned above are converted to boric acid in one way or another, boric acid is the bioavailable molecule uptaken from the soil by plants to nourish on boron. Depending on the environmental conditions (e.g., drought) and supplied concentration, boron fertilizers available on the market can easily convert deficiency into toxicity. According to the above, the boron fertilization is a common agricultural and silvicultural practice worldwide, but the known toxicity together with low solubility and complicated handling of boron fertilizers made necessary to find alternatives for them. For this reason, it is useful to develop new non-toxic products for both plants and humans which are capable of enhancing plant growth as an additional benefit.

Boronic acids are an alkyl or aryl substituted boric acid derivative containing a carbon-boron bond, and belonging to the large class of organoborons. Boronic acids act as Lewis acids, i.e., are chemical compounds that can accept electrons and are extremely important catalysts of certain organic reactions. Boronic acids are frequently used in organic chemistry as chemical synthesis components and intermediaries predominant in Suzuki cross-coupling. Boronic acids have not been used commercially as compound fertilizer. In 2004, Bassil E, Hu H, Brown PH. (2004) in its publication Use of phenylboronic acids to investigate boron function in plants. Possible role of boron in transvacuolar cytoplasmic strands and cell-to-wall adhesion. Plant Physiology 136:3383-95, reported the use of boronic acids in tobacco cell cultures as a tool to induce boron deficiency due to intracellular competition established by supplementing boron in different chemical forms. No test were conducted in plants to assess whether its effect induce boron deficiency much less to assess whether this element is supplemented for normal plant growth by boronic acids.

According to US 4,462,819 there is provided a unique fertilizer composition which comprises urea and/or a compound which is capable of producing urea when applied to the soil and a "urease inhibiting effective amount" of one or more organoboron acid compounds of the formula: R¹ R²BOH wherein R¹ and R² are the same or different and are hydroxy or substituted aryl radicals wherein substituents are one or more moieties selected from the group consisting of "electron donating groups", provided, however, that when either Ri or R₂ is hydroxy, the other is a substituted aliphatic or aryl radical, or one or more compounds which are capable of forming such acid compounds in situ. In this application the organoboron acid is used as urease inhibitor in the composition, i.e., it needs to be used together with urea, and is not used as boron fertilizer.

Unlike US 4,462,819, the present invention discloses the use of methylboronic acid of formula (CH₃B(OH)₂) as the chemical compound that supplements boron deficiency promoting leave and root growth and being not toxic for plants in high concentrations, methylboronic acid being one of the boronic acids that have a higher solubility simplifies handling and application.

Furthermore, animal cell studies have shown that methylboronic acid does not affect cell growth at in vitro culture conditions, making its use as fertilizer not harmful to human beings as indicated by Barranco, Kim DH, Stella SL Jr, Eckhert CD. (2009) Boric acid Inhibits stored Ca²⁺ release in DU-145 prostate cancer cells. Cell Biol Toxicol. 25:309-20. SOLUTION TO TECHNICAL PROBLEM

Boron fertilization is a common agricultural and silvicultural practice worldwide, but its known toxicity together with low solubility and difficult handling, made necessary to look for substitutes. There are several products on the market that are used for boron fertilization. These compounds are mainly based on boric acid, boron polyamines, boronatrocalcite and borax, among others. A high dose of soluble boron can produce toxic effects on plants, especially if the fertilizer containing the same is applied very close or highly concentrated, affecting their growth and yield. Despite boron importance for plants, the market for these fertilizers has been stagnant in recent years and new products have not been put on the market in the last 20 years. Boron deficiency is transient in many crops and is therefore difficult to prevent. Frequent treatments have been incorporated in many areas of the world to prevent deficiency, but these applications can become toxic due to environmental conditions and the narrow range of concentrations at which boron acts.

For this reason, it is useful to develop non-toxic boron fertilizer for plants that also is capable of deliver further benefits, such as enhancing the growth of plants introducing a differentiating factor over products already present on the market, which would be a great benefit to agricultural and silvicultural producers.

The present invention relates to a boron-based fertilizer, specifically with a powdered fertilizer comprising methylboronic acid and supplementing boron deficiency in plants. This powdered fertilizer has as comparative advantage being non- toxic to plants, enhancing its growth capacity, having higher water solubility and being not harmful to human cells.

SUMMARY OF INVENTION The present invention relates to a boron-based fertilizer, specifically with a powdered fertilizer comprising methylboronic acid, macronutrients and micronutrients.

Macronutrients include, but are not limited to, nitrate, ammonium, calcium, sulfate, phosphate, potassium and magnesium.

Micronutrients include, but are not limited to, chlorine, sodium, cobalt, copper, iron, iodine, molybdate and zinc.

This methylboronic acid powdered fertilizer is useful for supplementing boron deficiency increasing leave and root growth, on the other hand is highly soluble, so makes easy handling and application, is not toxic to plants when applied at high concentrations and innocuous to animal cells.

DESCRIPTION OF FIGURES

Figure 1 shows a graph of quantitative analysis of solubility of boric acid and boronic acids in water at 22 ^QC. BA: boric acid; MBA: methylboronic acid; PBA: phenylboronic acid; 3N-PBA: 3--nitrophenylboronic acid. The graph shows that MBA has at least 40% w/v of solubility; BA has 10% w/v of solubility; PBA and 3N-PBA has 5% w/v of solubility. It is clearly seen that MBA exhibits a higher solubility in water compared to boronic acid and other boronic acids analyzed. Figure 2 shows a graph quantifying main root length of plants grown in media supplemented with boron. Boron deficiency condition (deficit); plants supplemented with 6.2 mg/l boric acid (BA); plants supplemented with 6.2 mg/l methylboronic acid (MBA). Significant differences in main root length of plants treated with MBA and BA compared to boron deficiency at 7 days of treatment are observed. At 14 days significant differences in root length in plants treated with MBA compared with BA and boron deficiency condition were observed. In conclusion, MBA supplements boron deficiency and enhances growth for main root growth when its effect is compared to BA. The symbol ^* indicates significant differences. Figure 3 shows a graph quantifying lateral root number of plants grown in media supplemented with boron. Boron deficiency condition (deficit); plants supplemented with 6.2 mg/l boric acid (BA); plants supplemented with 6.2 mg/l methylboronic acid (MBA). At 14 days significant differences in lateral root number in MBA treated plants compared with BA and boron deficiency condition are observed. In conclusion, MBA supplements boron deficiency for lateral root growth and increased the number of lateral root. The symbol ^* indicates significant differences.

Figure 4 shows a graph quantifying in plant growth tests made with various boronic acids at in vitro culture conditions. 5 days old Arabidopsis thaliana plants were transferred to different growth media. Plants grown in medium supplemented with 6.2 mg/l methylboronic acid (MBA); plants grown in medium supplemented with 6.2 mg/l of phenylboronic acid (PBA); plants grown in medium supplemented with 6.2 mg/l uM of 3-nitrophenylboronic acid (3N- PBA). This figure shows the effect of various boronic acids on plant growth after 7 days of treatment.

Figure 4a shows a graph quantifying main root length; Figure 4b shows a graph quantifying lateral root number; Figure 4c shows a graph that depicts plant leave area in each treatment. PBA does not supplement the boron deficiency, plants show alterations in leave and root growth. No lateral roots and root hairs are observed. 3N-PBA is toxic for Arabidopsis thaliana at physiological concentrations. It is clearly shown that MBA is the only boronic acid analyzed that efficiently supplements boron deficiency. ^* Significant differences. Figure 5 shows a graph quantifying growth of root hairs and leaves on plants treated with methylboronic acid. Figure 5a shows a graph quantifying root hair length of plants grown in boric acid (BA) and plants grown in methylboronic acid (MBA); Figure 5b shows a graph quantifying leave area of plants grown in BA and plants grown in MBA. It is clearly seen that MBA enhances the growth and increases the number of root hairs and leaves. ^* Significant differences. Figure 6 shows a graph quantifying main root length of plants grown in media supplemented with toxic concentrations of boron. Control condition, culture medium supplemented with 6.2 mg/l of boric acid (BA) and methylboronic acid (MBA), respectively. Plants grown in culture medium supplemented with 310 mg/l of BA and MBA respectively. It is observed that at 14 days after treatment, high concentrations of boric acid significantly inhibited the growth of the main root compared to control condition. Moreover, it is observed that high concentrations of acid methylboronic did not inhibit the growth of main root, and it grows at the same length than in plants grown with 6.2 mg/l of MBA. In conclusion, a high dose of methylboronic acid is not toxic to plants.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention comprises a powdered fertilizer containing methylboronic acid (CH₃B(OH)₂) as boron nutrient and use thereof as a fertilizer either in powder form, dissolved in water or another solvent, for its application in soil fertilization, hydroponics, drip irrigation, foliar fertilization and flowers.

Said fertilizer is a powder for supplementing boron deficiency in plants, comprising: methylboronic cid, macronutrients and micronutrients, wherein methylboronic acid is from 0.14% to 7.41 % in said fertilizer; more specifically methylboronic acid is from 2.80% to 6.85% of said fertilizer.

In said powdered fertilizer, macronutrients comprise, but are not limited to,: from 42.87% to 37.07% of ammonium nitrate (NH₄NO₃); from 42.87% to 37.07% of potassium nitrate (KNO₃); from 5.72% to 6.18% of calcium chloride (CaCI₂); from 0.14% to 3.71 % of magnesium sulfate (MgSO₄) and from 5.72% to 6.18% of potassium phosphate monobasic (KH₂PO₄).

Similarly, in said fertilizer, micronutrients comprise, but are not limited to: from 0.03144% to 0.1236% of zinc sulfate (ZnSO₄); from 0.00009% to 0.0043% of copper sulfate (Cu₂SO₄); from 0.0018 to 0.0031 % of cobalt sulfate (CoSO₄); from 1 .43% to 0.3707% of manganese sulfate (MnSO₄); from 1 .072% to 1 .85% of ferrous sulfate (FeSO₄); from 0.0014% to 0.0124% of potassium iodide (Kl), and from 0.00715 to 0.0048% of sodium molybdate (Na₂MoO₄).

Powdered fertilizer to be used in solid form for direct soil application.

Furthermore, we have a fertilizer composition for supplementing boron deficiency comprising from 700 mg to 9,000 mg of powdered fertilizer diluted in one liter of water.

This fertilizer composition to be used in soil fertilization by regular irrigation, hydroponics, drip irrigation and foliar fertilization.

Methylboronic acid of powdered fertilizer delivers boron content similar to commercial fertilizers but exhibits higher solubility in water. Methylboronic acid delivers 16.9% of boron and has a solubility higher than 40% w/v when dissolved in water at room temperature (20-22^QC). Other boronic acids such as phenylboronic acid and 3-nitrophenylboronic acid deliver lower % of boron (8.2 and 6.3 respectively) and a maximum solubility of 5% w/v in both cases. Boric acid, the chemical absorbed by plants, delivers 16.3% of boron and has a solubility of 10% w/v, as shown in graph of Figure 1 .

Methylboronic acid in concentrations of at least 40% w/v is completely soluble in water, in contrast to boric acid, phenylboronic acid and 3- nitrophenylboronic acid. This higher solubility implies supplying higher boron content in smaller volume of water, which makes easier transport, handling and application.

Methylboronic acid supplements boron deficiency in plants. For assessing the ability of methylboronic acid as a boron fertilizer, assays of supplementation for this micronutrient deficiency in Arabidopsis thaliana plant were performed. Murashige and Skoog culture media were prepared at the recommended concentrations except for boron micronutrient. This micronutrient was replaced by boric acid and methylboronic acid at a concentration recommended for this plant (6.2 mg/l) and using boron deficiency conditions as control. The boron deficiency condition is defined as the culture medium containing all the macro- and micronutrients except boron element. In the graphs of Figures 2 and 3 it can be seen that methylboronic acid is capable of supplementing boron deficiency by quantify main root length and lateral root number.

Methylboronic acid is a boronic acid for supplementing boron deficiency. It was assessed if other boronic acids had the ability to fertilize with boron in Arabidopsis thaliana plants. As shown in the graph of Figure 4, the methylboronic acid is the only of all boronic acids assessed that supplements boron deficiency in this plant. The use of phenylboronic acid do not efficiently supplement boron deficiency. Plants had an abnormal growth of leaves and roots. No lateral roots and root hairs were observed. Moreover, the use of 3- nitrophenylboronic acid was toxic for Arabidopsis thaliana at physiological concentrations, so use thereof in boron fertilizer composition is not useful because small doses kills plants

Methylboronic acid enhances plant growth. Methylboronic acid effect on leave and root growth was evaluated and compared with the effect of boric acid, which is the chemical compound used in commercial boron fertilizers. In the graphs of Figures 2 and 3 is shown quantitatively that methylboronic acid has a greater increase in the number of main and lateral roots. Furthermore, as shown in the graph of Figure 5, methylboronic acid treatment increased the number of root hairs and leaf size. Therefore, the use of methylboronic acid as boron fertilizer enhances plant growth, promoting an increase in roots and leaves.

Methylboronic acid is not toxic for plants. The current boron fertilizers are toxic in high concentrations. Assays at high concentrations of boric acid and methylboronic acid (310 mg/l) were performed to assess if methylboronic acid is toxic. In the graph of Figure 6 can be seen that high concentrations of methylboronic acid had no significant effects on Arabidopsis thaliana growth, when comparing low with high concentrations, allowing normal growth of roots which are direct target of boron toxic effect. APPLICATION EXAMPLES

EXAMPLE 1 . Methylboronic acid supplements boron deficiency. In vitro tests using Arabidopsis thaliana were performed. For this purpose plants were grown for 5 days and were then transferred to various media with the following fertilizer composition comprising: macronutrients (1 ,900 mg/l potassium nitrate; 1 ,650 mg/l ammonium nitrate; 370 mg/l magnesium sulfate, 170 mg/l potassium phosphate monobasic, 440 mg/l calcium chloride), micronutrients (8.6 mg/l zinc sulfate, 0.025 mg/l copper sulfate; 0.025 mg/l cobalt sulfate; 0.25 mg/l sodium molybdate; 22.30 mg/l manganese sulfate, 0.83 mg/l potassium iodide; 27.8 mg/l ferrous sulphate), organic additives (0.5 mg/l acid nicotinic, 0.5 mg/l pyridoxine, 0.1 mg/l thiamine, 100 mg/l myo-inositol) and phytagel agar 8 g/l at pH 5.8. Boron micronutrient was supplement in the form of boric acid and in a first assay and it was supplemented in the form of methylboronic acid in a second assay, both at a concentration of 3 to 6.2 mg/l. Water was used as solvent.

Evaluations at 7 and 14 days after treatment were performed. Plants grown in media without supplemental boron, deficiency condition, were used as control. Boron deficiency inhibits plant growth, of both root and leaves. From this example is observed that methylboronic acid supplements boron deficiency, wherein root and leave growth is higher than plant grown in boric acid supplemented medium. When quantifying parameters such as main root length and lateral root number, it can be observed that methylboronic acid enhances plant growth (graphs of Figures 2 and 3). The same is observed when viewing in detail root hair number and leave size (graph in Figure 5). In conclusion, methylboronic acid supplements boron deficiency, it may then be used in the development of fertilizers and also enhances plant growth. EXAMPLE 2. Methylboronic acid is not toxic to plants at high concentrations.

It is known that high boron concentrations are toxic to plants. In vitro assays to evaluate the toxicity of methylboronic acid were performed using Arabidopsis thaliana. For this purpose plants were grown for 5 days and were then transferred to various media containing: Macronutrients Macronutrients (1 ,900 mg/l potassium nitrate; 1 ,650 mg/l ammonium nitrate; 370 mg/l magnesium sulfate, 170 mg/l potassium phosphate monobasic, 440 mg/l calcium chloride), micronutrients (8.6 mg/l zinc sulfate, 0.025 mg/l copper sulfate ; 0.025 mg/l cobalt sulfate; 0.25 mg/l sodium molybdate; 22.30 mg/l manganese sulfate, 0.83 mg/l potassium iodide; 27.8 mg/l ferrous sulphate), organic additives (0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine, 0.1 mg/l thiamine, 100 mg/l myo-inositol) and agar supplemented with 310 mg/l of boric acid or methylboronic acid. Plant growth was observed during 14 days after treatment. It was observed that boric acid was highly toxic to the plant, inhibiting the growth of main and lateral root besides withering leaves. In contrast, plants grown at high concentrations of methylboronic acid did not show major alterations in plant growth, main root sustained its growth (see graph in Figure 6) and leaves are observed to have green color, similar to control plant.

EXAMPLE 3. Methylboronic acid supplements boron deficiency compared to other boronic acids.

Boronic acids are aryl or alkyl substituted boric acid derivatives. We assessed whether other boronic acids had the ability of supplementing boron deficiency. For this purpose plants were grown for 5 days and were then transferred to various media containing: macronutrients (1 ,900 mg/l potassium nitrate; 1 ,650 mg/l ammonium nitrate; 370 mg/l magnesium sulfate, 170 mg/l potassium phosphate monobasic, 440 mg/l calcium chloride), micronutrients (8.6 mg/l zinc sulfate, 0.025 mg/l copper sulfate ; 0.025 mg/l cobalt sulfate; 0.25 mg/l sodium molybdate; 22.30 mg/l manganese sulfate, 0.83 mg/l potassium iodide; 27.8 mg/l ferrous sulphate), organic additives (0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine, 0.1 mg/l thiamine, 100 mg/l myo-inositol) and 8 g/l Phytagel agar at pH 5.8. These media were supplemented with 6.2 mg/l of methylboronic acid, phenylboronic acid and 3-nitrophenylboronic acid. Plant growth was observed at 7 days after treatment. It was observed that 3-nitrophenylboronic acid was toxic, completely inhibiting leave and root growth. Moreover, phenylboronic acid did not supplement boron deficiency, and plants had alterations in leave and root growth. No lateral roots and root hairs were observed. It is noted that methylboronic acid is the only one of the boronic acids analyzed that efficiently supplemented boron deficiency by quantifying main root length, lateral root number and leave size parameters (see graph in Figure 4).

EXAMPLE 4. Methylboronic acid is highly soluble in water. The market for new boron fertilizer is directed to development of products in liquid form. Methylboronic acid was dissolved in water at room temperature (20-22 ^QC) to assess its soluble ability, comparing its ability to boric acid, and other boronic acids. As shown in the graph of Figure 1 , methylboronic acid has a solubility of at least 40% w/v, higher ability that the one of boric acid (10%) and other boronic acids (5%). This high solubility favors the use of methylboronic acid in developing liquid boron fertilizers.

Claims

1 . Powdered fertilizer for supplementing boron deficiency in plants comprising: methylboronic acid, macronutrients and micronutrients.

2. Powdered fertilizer according to claim 1 , wherein the acid methylboronic acid is from 0.14% to 7.41 %.

3. Powdered fertilizer according to claim 1 , wherein methylboronic acid is from 2.80% to 6.85%.

4. Powdered fertilizer according to claims 1 to 3, wherein macronutrients are: from 42.87% to 37.07% of ammonium nitrate; from 42.87% to 37.07% of potassium nitrate; from 5.72% to 6.18% of calcium chloride; from 0.14% to 3.71 % of magnesium sulfate and from 5.72% to 6.18% of potassium phosphate monobasic.

5. Powdered fertilizer according to claims 1 to 4, wherein micronutrients are: from 0.03144% to 0.1236% of zinc sulfate; from 0.00009% to 0.0043% of copper sulfate; from 0.0018 to 0.0031 % of cobalt sulfate; from 1 .43% to 0.3707% of manganese sulfate; to 1 .072% to 1 .85% of ferrous sulfate; from 0.0014% to 0.0124% of potassium iodide and from 0.00715 to 0.0048% of sodium molybdate.

6. Use of the powdered fertilizer described in claims 1 to 5, to be used in solid form for direct soil application.

7. Fertilizer composition for supplementing boron deficiency in plants, comprising from 700 mg to 9,000 mg of the powdered fertilizer described in claims 1 to 5 diluted in one liter of water.

8. Use of the fertilizer composition described in claim 7, to be used in soil fertilization by regular irrigation, hydroponics, drip irrigation and foliar fertilization.