Method of treating maize seeds to improve growth, seeds treated by this method, and use thereof

Abstract

The present invention relates to a method of treating maize seeds to improve growth, which comprises the steps of: cultivating the yeast strain Rhodotorula mucilaginosa CCY 20-1-39 for 3 to 5 days in darkness at a temperature in the range of 26 to 30 °C; applying the culture of the yeast strain Rhodotorula mucilaginosa CCY 20-1-39, at a concentration of 0.5 to 1.5 x 108 yeast cells per 1 ml of aqueous suspension, to maize seeds in a ratio of at least 90 ml of suspension per 150 g of maize seeds, preferably 100 ml of suspension per 150 g of maize seeds; and exposing the prepared mixture from the preceding step to a continuous homogeneous oxygen supply for 2.5 to 3.5 hours.

Description

[0001] Method of treating maize seeds to improve growth, seeds treated by this method, and use thereof

[0002] Technical Field

[0003] The invention relates to the field of agriculture, in particular the use of yeast to improve plant growth under various conditions.

[0004] Background Art

[0005] Drought and climate change have caused significant crop losses of agriculturally important plants in recent years. Their impact causes damage to plants, deterioration of their overall health, and reduced production. With worsening conditions, production costs are rising and crop yields are insufficient, which has a negative economic impact on farmers. To boost plant production under normal / natural conditions, as well as during periods of low rainfall or extreme weather, natural substances known as biostimulants are increasingly being used on a large scale.

[0006] Currently, the challenge of improving fertility, whether under normal / natural conditions or drought, is being addressed by cultivating more resistant cultivars or adjusting agrotechnical practices, however, these approaches are not always successful and are often more demanding technically and economically. Biostimulants are most commonly used in the form of extracts from algae and organic substances (humic acids).

[0007] In Slovakia, the use of biostimulants is becoming increasingly popular, especially in the context of sustainable agriculture and organic farming. Farmers in Slovakia are beginning to integrate biostimulants and other growth promoters into their cultivation practices, especially in the areas of vegetable and fruit growing and viticulture. Biostimulants are often used in combination with traditional fertilizers and pesticides (used in lower concentrations), thereby increasing overall efficiency and reducing negative environmental impacts. These biostimulants are mostly used in the form of sprays / watering. Mycorrhizal fungi are used as biostimulants and are applied to the soil on a carrier medium, such as sand or sawdust, into which they are incorporated. Plants grown from seeds treated with these biostimulants are better able to develop and produce yields, even under conditions of low rainfall.

[0008] Summary of the Invention

[0009] The inventors of this invention found that yeast has the potential to positively influence the health and yield of plants not only under normal / natural conditions, but also under stress conditions.

[0010] The subject-matter of this invention is a method of treating maize seeds before sowing, by applying live Rhodotorula mucilaginosa yeast cells, thereby improving seed germination. The application of this yeast is effective in improving plant growth both under normal / natural conditions and under conditions of reduced rainfall.

[0011] - An advantage of the yeast applied in this manner is that it can be applied directly before sowing to different maize hybrids, so that storage of the treated seeds for a longer period of time is not required.

[0012] - Application of the yeast in the form of a suspension to the seeds provides not only the yeast cells themselves but also liquid (water), which is essential for seed imbibition. During this process, metabolic activities are activated in the seeds that ensure germination.

[0013] - An advantage is that the yeast can accelerate seed imbibition (by releasing various extracellular substances) and subsequently facilitate the emergence of the seedling through the seed coats.

[0014] - A further advantage is that, unlike other commonly used biofertilizers or biostimulants which must be applied repeatedly, a single application of the yeast Rhodotorula mucilaginosa CCY 20-1-39 can improve yield both under normal / natural conditions and under adverse conditions. The method of treating maize seeds to improve growth according to this invention comprises the steps of:

[0015] - cultivating the yeast strain Rhodotorula mucilaginosa CCY 20-1-39 for 3 to 5 days in darkness at a temperature in the range of 26 to 30 °C;

[0016] - applying the culture of the yeast strain Rhodotorula mucilaginosa CCY 20-1- 39, at a concentration of 0.5 to 1.5 x 108yeast cells per 1 ml of aqueous suspension, to the maize seeds in a ratio of at least 90 ml of suspension per at least 150 g of maize seeds, preferably 100 ml of suspension per 150 g of maize seeds;

[0017] - exposing the prepared mixture to continuous homogeneous oxygen supply for 2.5 to 3.5 hours.

[0018] Wherein the suspension is preferably composed of the yeast strain Rhodotorula mucilaginosa CCY 20-1-39 and water.

[0019] Preferably, the water may be water from commonly available sources, distilled water, redistilled water, or sterile water.

[0020] Where the ratio of the suspension is preferably such that the whole maize seeds are completely immersed in the suspension.

[0021] Preferably, Luciana hybrid maize seeds are used, but other silage hybrids can also be used, e.g. Markiza, ES FIREBALL, ES LEGOLAS, MASTODON.

[0022] According to a preferred embodiment, sufficient homogeneous oxygen supply is ensured by mixing the mixture at 35-45 rpm, preferably at 40 rpm in air.

[0023] Preferably, a shaker may be used for the mixing.

[0024] This ensures an adequate amount of oxygen for the yeast and seeds, as well as uniform coverage of the seeds with the yeast suspension.

[0025] The findings obtained have potential for application in plant production. According to a preferred embodiment, the cultivation of the yeast strain is carried out for 4 days.

[0026] According to another preferred embodiment, the cultivation of the yeast strain is carried out at a temperature of 28 °C.

[0027] According to a further preferred embodiment, the application of the yeast strain is carried out at a concentration of 1 x 108yeast cells per 1 ml of suspension.

[0028] Another aspect of this invention is a maize seed treated by the method described herein.

[0029] Another aspect of the invention is the use of the yeast strain Rhodotorula mucilaginosa CCY 20-1-39 for treating maize seeds to improve growth.

[0030] The yeast Rhodotorula mucilaginosa is deposited with the Culture Collection of Yeasts (acronym CCY) at the Institute of Chemistry of the Slovak Academy of Sciences, v. v. i., Bratislava, under accession number CCY 20-1-39, with the date of deposit 30 November 2014. This strain was isolated by staff of the Culture Collection of Yeasts from soil beneath an apricot tree (Prunus armeniaca) \ \ Male Leva re, Slovakia, in 2013.

[0031] This yeast strain can interact with soil particles as well as with other organisms present in the soil, thereby influencing the availability of nutrients, water, and also air for plant organs (roots). The properties of yeasts isolated from different environments may be strain-specific; for example, they may have different optimal growth temperatures, nutritional and pH requirements, tolerance to chemical substances, etc. Likewise, the extracellular substances that they produce for their own protection against adverse conditions, as well as for the protection of other organisms, may differ in composition. The yeasts applied to maize seeds prior to sowing act as a biostimulant for improving plant growth under natural conditions (sufficient irrigation) and under drought stress conditions.

[0032] It has been found that the strain Rhodotorula mucilaginosa isolated from the soil is more tolerant to various stress factors characteristic of soil, such as prolonged drought, prolonged low or high temperatures, fluctuating availability of nutrients and oxygen, and the presence of various chemicals in the soil. The use of this strain in the method according to the present invention ensures higher yield and better growth of cultivated crops, especially maize, compared to strains isolated from the aerial parts of plants.

[0033] Further advantages of the yeast R. mucilaginosa CCY 20-1-39 are its simple cultivation and non-pathogenicity. It exhibits high adhesion and is capable of covering the entire surface of the plant seed.

[0034] An advantage of the method of using the yeast according to the present invention is its simplicity, as well as the uniform application of the yeast to maize seeds, thereby ensuring more effective utilization of the biostimulant after sowing maize at a given site. The method is energy-efficient and inexpensive to prepare.

[0035] Detailed description

[0036] Methodology and Research Results

[0037] Maize seeds (Luciana hybrid) purchased from RWA SLOVAKIA spol. s.r.o. were used for testing. This hybrid is more sensitive to drought compared to other maize hybrids available from this supplier. For experiments aimed at monitoring plant growth after application of the yeast to the seeds, a universal horticultural substrate Durpeta, purchased from the company ZRT, spol. s r. o., was used.

[0038] Example 1 Protocol for cultivation of maize plants with the optimal yeast concentration

[0039] - The yeast strain Rhodotorula mucilaginosa, CCY 20-1-39) was cultivated on wort agar for 4 days in the dark at a temperature of 28 °C;

[0040] - From the four-day-old yeast cultures, an aqueous suspension was prepared at a concentration of 1 x 108yeast cells per 1 ml of suspension;

[0041] - The suspension of live yeast cells was applied to maize seeds (hybrid Luciana) for three hours (during which imbibition of the seeds occurred) under continuous mixing on a shaker at 40 rpm for 3 hours; the ratio used was 100 ml of suspension per 150 g of maize seeds, and the maize seeds were completely immersed in the suspension;

[0042] - The treated seeds were subsequently transferred into large Petri dishes, where they were allowed to germinate in darkness for 45 hours. During this step no further mixing on the shaker was performed (this pregermination step was carried out in order to maintain the same number of germinated seeds in each experimental variant under comparison: control, yeast, drought);

[0043] - After germination, the seeds were planted in horticultural substrate in pots (each pot contained 5 litres of substrate and 4 maize seeds treated with the yeast);

[0044] - The horticultural substrate was mixed with water to achieve soil moisture capacity (70% for normal / natural conditions and 35% for drought conditions);

[0045] - To maintain either normal / natural conditions or drought conditions, the gravimetric method was used— regular weighing (every second day) and adjustment of soil moisture so that throughout the experiment the soil moisture remained the same as at the beginning;

[0046] - The following growth conditions were maintained: temperature 25 / 20 °C, photoperiod 16 h light / 8 h dark, light intensity 350 pmol photons irr^s-1, and 40% air humidity;

[0047] - Evaluation of the experiment was performed after three weeks of plant growth (plants with 3 fully developed true leaves and a fourth developing leaf) by determining shoot length, fresh and dry weight of the shoot, and leaf area of the third leaf. Example 2

[0048] In this example, the protocol described in Example 1 was used with different concentrations, namely 105yeast cells / ml of suspension, 107yeast cells / ml of suspension, 108yeast cells / ml of suspension, and 109yeast cells / ml of suspension. In the horticultural substrate, a soil moisture capacity of 70% was maintained for normal / natural conditions throughout the entire duration of the experiment. The other parameters were kept the same as in Example 1. After three weeks of growth, the following growth parameters were evaluated: shoot length, fresh and dry weight of the shoot, and leaf area of the third leaf. The results are shown in Table 1. Maize without yeast application was used as the control.

[0049] Table 1

[0050] The results showed that the application of yeast cells at a concentration of 108yeast cells per 1 ml of suspension had a stimulatory effect on the growth of maize plants under normal / natural conditions. A statistically significant difference compared with the control is highlighted.

[0051] Indices a and b indicate statistical differences between individual treatments at P < 0.05, where the P value represents the level of significance, indicating that with less than 5% probability the datasets are not different, respectively, with more than 95% probability the datasets are different.

[0052] Example 3 In this example, the protocol from Example 1 and the same concentrations of applied yeast cells as in Example 2 were used. In the horticultural substrate, a soil moisture capacity of 35% was maintained for drought conditions throughout the entire duration of the experiment. The results are shown in Table 2. Maize without yeast application under drought conditions was used as the control.

[0053] Table 2 The results showed that the application of yeast cells at a concentration of 108yeast cells per 1 ml of suspension had a stimulatory effect on the growth of maize plants under drought conditions. A statistically significant difference compared with the drought control is highlighted. The indices a, b, and c indicate statistical differences between the individual treatments at P < 0.05, where the P value represents the significance level, indicating that with less than 5% probability the datasets are not different, respectively, with more than 95% probability the datasets are different.

Claims

Claims1. A method of treating maize seeds to improve growth, characterized in that it comprises the steps of: a) cultivating the yeast strain Rhodotorula mucilaginosa CCY 20-1-39 for 3 to 5 days in darkness at a temperature in the range of 26 to 30 °C; b) applying the culture of the yeast strain Rhodotorula mucilaginosa CCY 20-1- 39, at a concentration of 0.5 to 1.5 x 108yeast cells per 1 ml of aqueous suspension, to maize seeds in a ratio of at least 90 ml of the suspension per 150 g of maize seeds, preferably 100 ml of the suspension per 150 g of maize seeds; c) exposing the prepared mixture from step b) to continuous homogeneous oxygen supply for 2.5 to 3.5 hours.

2. The method of treating maize seeds to improve growth, according to claim 1, characterized in that, the step c) is carried out by mixing in air at 35-45 rpm, preferably at 40 rpm in air.

3. The method of treating maize seeds to improve growth, according to claim 1 or 2, characterized in that the cultivation in the step a) is carried out for 4 days.

4. The method of treating maize seeds to improve growth, according to claim 1, 2, or 3, characterized in that, the cultivation in the step a) is carried out at a temperature of 28 °C.

5. The method of treating maize seeds to improve growth, according to any one of the preceding claims, characterized in that, the concentration of the yeast strain in the step b) is 1 x 108yeast cells per 1 ml of suspension.

6. A maize seed treated by the method according to any one of the preceding claims.

7. Use of the yeast strain Rhodotorula mucilaginosa CCY 20-1-39 for treating maize seeds to improve growth.

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