CZ32478U1 - Biological agent for post-harvest protection of fruits and / or vegetables - Google Patents

Description

The Industrial Property Office does not ascertain in the registration procedure whether the subject of the utility model meets the conditions of eligibility for protection pursuant to Section 1 of Act no. E. 478/1992 Coll.

Biological agent for post-harvest protection of fruits and / or vegetables

Technical field

The invention relates to a biological composition for post-harvest protection of fruit and / or vegetables

BACKGROUND OF THE INVENTION

Technologies for the storage of fruit and vegetables and their protection against microbial pests have progressed significantly in recent years. Today, they are based on the combined use of physical, chemical and biological factors. The most commonly used physical factors include a reduced temperature (usually around 2 to 4 ° C) and a regulated, often reduced level of moisture applied in warehouses. Chemical antifungal protection includes the use of nitrogen-free inert atmosphere (nitrogen generators in large-scale warehouses), the use of other volatile substances applied simultaneously with the nitrogen atmosphere (volatile ripening regulators) and, last but not least, chemical fungicides providing still primary protection against fungal pests for fruit and vegetables during their storage. These are public concerns about the possibility of contamination of fruit and vegetables with chemical residues as well as the possibility of breeding strains of mold resistant to the chemicals used.

Particularly stringent legislation is introduced in the EC conditions, even leading to a ban on the use of certain chemicals if their negative effects on food quality or human health are suspected.

For the above reasons, a number of non-chemical processes have been proposed for the preservation of fruit and vegetables using natural substances such as flavorings (for example, "hot"), acetic acid, phosphoric acid, jasmonates, glycosinolate, propolis, fusapyron, deoxyfusapyron, chitosan , essential oils or plant extracts. A disadvantage of these natural substances in the preservation of fruit and vegetables during storage is their variable effect, which has hitherto prevented their widespread use in storage technologies (Tripathy P., Dubey NK / 2004 / Postharvest Biol Technol 32, 235-245).

Biological control means in the form of bacterial or fungal antagonists of economically important fungi represent another possibility for the natural protection of fruits and vegetables during storage. The action of these microorganisms is most often based on biological antagonism, production of antibiotic-like substances, spatial and nutritional competition, or the recently intensively studied process of induction of plant immunity. In practice, about fifty different types of microorganisms are already used, such as Acremonium brevae, Aureobasidiiim pullulans, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Biirkholderia cepacia, Brevundimonas diminuta, Candida guilliermondii, Candida sangranifaciens. species (Sharma RR, Singh D, Singh R / 2009 / Biol Control 50, 205-221). Mention may also be made of specific products already marketed and marketed of this type, such as the AQ-10 biofungicide of Ecogen Inc. containing Ampelomyces quisqualis for the protection of apples, grapes, strawberries, tomatoes and cucumbers, Aspire of the same company based on Candida oleophila strain 1-182, Biosave 10 LP from Eco Science Corp., containing Pseudomonas syringae and many others. Although the number of microorganisms practically used seems almost inexhaustible, the criteria for a suitable candidate set by professional firms are in fact quite strict. These include in particular (a) genetic stability, (b) good efficacy even at low concentrations, (c) very selective nutritional requirements, (d) ability to survive in adverse environments, (e) efficacy against as wide a spectrum of pathogens as possible. a large number of different agricultural commodities; (f) resistance to common chemical pesticides;

U1 metabolites harmful or potentially harmful to humans, (h) not be pathogenic to the host (protected crop), (i) easy to prepare in a form suitable for storage and application, (j) compatible with the wide range of physical and chemical uses used conditions. In practically applied formulations, microbiological agents are administered in the form of various compositions, and it is known that adjuvants in these compositions may in some cases also affect the efficacy of the biological composition.

Post-harvest protection of fruit and vegetables is also the subject of a large number of patents and patent applications, although their number is far below the number of patents for biopesticide plant protection products per se. This also reflects the above-mentioned situation in the patent area, where chemical mixtures usually have a wider range of pests and protected crops compared to biological pesticides. As an example of several hundred patents filed for harvest protection, patent EP 2245934 B1, owned by BAYER CROPSIENCE AG, for a compound fungicide, effective on 28 species of fungi, representing the spectrum of the most common pathogens of post-harvest fungal diseases vegetables. GB 2541907 And Applicant AGRONATURALIS Ltd. discloses a fungicidal composition for the elimination of fungal pathogens producing mycotoxins. Other patents disclose devices suitable for use in post-harvest fruit and vegetable protection, or a combination describing agents and methods of application for post-harvest protection. An example of a method, apparatus and use for post-harvest protection is US 7449206 B2 describing a device suitable for post-harvest protection of beans, or JP 2017/165654 A describing an agent and method of application for post-harvest disease control. Also of interest is EP 2503892 B1, the Applicant of the present invention, directed to the use of Pythium oligandrum as an active agent in combination with an inorganic or organic carrier to prevent and eliminate pathogenic agents causing post-harvest loss microbial or post-harvest application. protected crops include namely fruits, vegetables, nuts, cereals and other crops.

The essence of the technical solution

These disadvantages are overcome or substantially reduced by the biological composition for post-crop protection of fruits and / or vegetables by fumigation according to the present invention, which comprises a mixture of 2.0 to 4.0 wt. a biological fungicide; 3.34 to 6.6 wt. glycerol; 0.06 to 0.14 wt. calcium chloride dihydrate; and the remainder to 100 wt. water. The biological fungicide contains up to 90 wt. % silica and the remainder to 100 wt. of a biological substrate with a Pythium oligandrum M1 microorganism, wherein the Pythium oligandrum M1 microorganism has an oospore content of (1.0 ± 0.1) x 10 ⁶ per g of the fungicide. The desired oospore content is standardized with silica and Pythium oligandrum is grown on the substrate during application. The ground substrate may be millet and / or maize, a residual residue in the manufacture of Pythium oligandrum. The ground substrate facilitates the application of the biological mixture in the post-harvest application.

Food grade glycerol and distilled water can be used to enhance the quality of the biological composition. Glycerol is a dispersing agent in the mixture. The amount of water in the mixture has been tested to be optimal for the application of the biological post-harvest biological composition to fruits and vegetables.

It is preferred that the biological composition containing 100-200 g of the biological fungicide as defined above is mixed with water to a volume of 5 liters of the application biological composition. This creates an aqueous suspension suitable for application to 500 m ^{3 of} storage space with fruit and / or vegetables.

-2GB 32478 U1

The main advantage of the biological composition according to the present invention is the surprising finding that application for the protection of fruits and / or vegetables after harvesting and storage can be carried out by a conventional fumigation method using a very low concentration of biological fungicide with Pythium oligandrum. The use of this biological composition according to the invention after harvesting and in the storage of fruit and / or vegetables results in a very effective protection against molds, also associated with the possibility of prolonged storage of fruit and vegetables, which can be carried out at reduced and even normal temperature without use nitrogen or other inert inert atmosphere. The degree of protection using this biological agent is dependent on the condition of the fruits at the time of harvest, with the fact that the mildew affected by the fungus cannot be protected at the time of harvest. At the same time, however, it has been observed in several fruits and vegetables that there is no further spread of fungal infection from the affected fruit to the neighboring fruit, which is thus protected from the fungal attack. This aspect is very important because the affected fruits can be easily separated from other fruits and discarded while physically adjacent fruits are not damaged.

A particular advantage of the biological composition according to the present invention applied by conventional fumigation technology is the possibility of its efficient application in a large space using fumigated chilled microparticles, where it is normally possible to effectively treat up to 70 tons of stored fruit or vegetables in warehouses of up to 500 m ³ . Thus, the present biological composition according to the present invention is also applicable on an industrial scale for modern warehouses in which it can become part of other technologies used. Application of claimed biological agents in warehouses using fogging technology, resp. other technologies working in vapor is the preferred process (low technical demands and high efficiency).

In post-harvest application, the biological agent can now be very advantageously applied after storing the fruit in the refrigeration chambers of the warehouses or warehouses by the fumigation method, whereby the composition is dispersed in the form of fog by means of special biofumigators. allows a very effective application of the biological agent to the individual protected fruit. This methodology is also referred to as fogging. An important condition here is not only the general condition of cooling the mouth of the fumigator with water to a temperature compatible with the application of the biological agent, but also the condition that all substances necessary for proper dispersion and subsequent functioning of the biological agent are present in the application mixture.

For fruits and vegetables coming directly from the warehouses to the consumer's tables, it is not possible to apply most post-harvest chemical products for toxicological and regulatory reasons, while the defined biological fungicide used in the described biological composition is considered completely non-toxic and safe. The advantage is its safety and application of relatively very small amounts of biological agent.

Another advantage of the biological composition according to the invention is the flexibility of the defined biological fungicide in terms of application. After many years of development, the preparation of a defined biological fungicide has been refined to such an extent that it can now be produced in the form of a very fine powder, which can be easily prepared by slurrying in an aqueous medium a very small particle size suspension. The suspensions thus formed, especially in mixtures using glycerol as a dispersing agent, contain particles of a size which can be applied by means of most spraying devices with conventional filters having a mesh size of 0.2 mm or more.

-3 GB 32478 U1

Examples of technical solutions

As part of the cooperation of the applicants of this technical solution with the Polish partner by BioAgris and mainly due to independent research activities of both applicants, the amount of defined biological fungicide Polyversum containing the microorganism Pythium oligandrum in the application biological agent according to this technical solution was set to 100 to 200 g of a biological fungicide, preferably 150 g, mixed with water to a volume of 5 liters of water and applied to a 500 m ³ storage area with fruit and / or vegetables.

Further detailed research by the Applicants has revealed that it may be advantageous for individual crops to prepare application mixtures of somewhat different compositions and dosages, as further shown in Table 1.

In the case of soft crops such as strawberries and sour cherries, it has proved to be advantageous to reduce the amount of biological fungicide somewhat in the biological composition (and proportionally also reduce the amount of auxiliary components - calcium chloride and glycerol). silicon dioxide (components of biofungicide) and glycerol, the presence of which on fruits may cause problems in their storage and subsequent sale.

Conversely, in the case of crops in which peeling (e.g. cabbage) is peeled prior to sale, higher concentrations of the biological fungicide than the initial reference value may be used to increase efficacy.

The determination of the content of distilled water and calcium chloride dihydrate was determined to be optimal and preferred; especially if distilled water is not used, due to the well-defined tap water composition.

In Table 1, the content of the components, ie water, biofungicide, calcium chloride dihydrate and glycerol, is always given in% by weight.

In Table 1, the last column, representing the amount of biofungicide per 500 m ^{3 of} storage area, is given in g.

In all exemplary embodiments, the Pythium oligandrum M1 microorganism was standardized to an oospore content of (1.0 ± 0.1) x 10 ⁶ per g defined Polyversum biological fungicide.

In exemplary non-limiting embodiments, the biological composition for post-harvest protection of apples, strawberries, sour cherries and cabbage is described in detail.

-4GB 32478 U1

Table 1

Summary of the percentage of components in the biological agent and the total amount of biological fungicide applied to 500 m ³ storage areas

Crop Distilled water Biofungicide containing Pythium oligandrum Calcium chloride dihydrate Food grade glycerol Amount of biofungicide per 500 m ^{3 of} storage area Apples 91.9% 3.0% 0.1% 5% 150 g Strawberries 94.6% 2.0% 0,06% 3.34% 100 g Cherries 93.52% 2.4% 0.08% 4.0% 120 g Cabbage 89.26% 4.0% 0.14% 6.6% 200 g

Example 1

Combined pre-harvest and post-harvest apple treatment

In cooperation with Polish company BioAgris, business partner of Biopreparáty spol. s r. o., experiments with combined pre-harvest and post-harvest apple protection using the defined biological fungicide Polyversum were carried out in coordination with planters in Poland and the Czech Republic.

This Polyversum preparation was applied in pre-harvest mode in accordance with patent EP 2503892 B1 of Biopreparáty spol. s r. o., following the procedure described in the instructions for the authorized product in an amount of 100 g per 1 ha.

It was then applied in post-harvest protection by a conventional method of fumigating the application biological agent according to the invention in an amount of 150 g in 5 liters per 500 m ^{3 of} storage area. Pre-harvest application was performed using a standard field sprayer with a mesh size of 0.2 mm (90 mesh in technical marking).

In the post-harvest mode, the application by the biological agent according to the technical solution was carried out by means of Pulsfog Bio K22 fumigators.

Distilled water was used in the cooling vessel and the composition shown in Table 1 by weight was used in the biological agent container. %; in particular: 150 g of biological fungicide were mixed with 250 g of food grade glycerol, 4595 g of distilled water and 5 g of calcium chloride dihydrate.

Control apples were obtained from local growers or from markets (under normal spray with chemical antifungal 4 weeks and 2 weeks before harvest) and were treated in a standard regime according to the methodology used. All samples were placed in separate cooling boxes at 4 ° C during the first day after collection, fumigated and then the first set of samples was collected. Subsequently, samples were taken after 1, 2 and 3 months of storage and a visual assessment of the condition of 100 apples and a microbiological evaluation of the culture of 100 selected samples.

The results showed that both pre-harvest treatment and post-harvest fumigation had a significant effect on the level of fungal infection detected by culture microbiology: whereas in control apples, fungi were found in 42% of analyzed apples after 4 weeks of storage, this value decreased to 10%.

If the apples were also pre-harvested by spraying, these values were 9% and 3% respectively. The results also showed a practically significant result of the evaluation of the condition of apples on a sample of 1000 fruits.

When apples were sprayed with a Polyversum biological composition containing 75 to 90% by weight of silica and 10 to 25% by weight of ground substrate with growth of Pythium oligandrum M1 pre-harvest and post-harvest, only 3 months of storage in the refrigerator at 4 ° C the percentage of decayed fruit, while the untreated control was essentially at least 30% decayed at that time. In addition, a very interesting finding was obtained that the treated apples did not spread the mold from the affected to the treated, and even slightly affected fruits (minor abrasions during transport) were not rotting or perishable.

Example 2

Combined pre-harvest and post-harvest strawberry treatment

In cooperation with BioAgris, business partner of Biopreparáty spol. s r. o., experiments were performed on strawberry plantations in Poland and the Czech Republic with combined pre-harvest and post-harvest strawberry protection using the biological fungicide Polyversum. This preparation was applied in the pre-harvest regime in accordance with the patent EP 2503892 B1 of the owner Biopreparáty spol. s r. o., following the procedure described in the instructions for the authorized product in an amount of 100 g per 1 ha. Pre-harvest application was performed using a standard field sprayer with a mesh size of 0.2 mm (90 mesh in technical marking).

In post-harvest protection, the apples were treated by fumigating the application biological agent at a rate of 150 g in 5 liters of aqueous suspension per 500 m ^{3 of} storage area. In the post-harvest mode, the biological agent according to the present invention was applied using Pulsfog Bio K22 or Pulsfog Bio K30 fumigators, using distilled water in a secondary cooling vessel. In the tertiary vessel, a biological composition having a composition in wt. % according to Table 1. In a particular case, 100 g of Polyversum biological fungicide were mixed with 167 g of food grade glycerol, 4730 g of distilled water and 3 g of calcium chloride dihydrate.

Control strawberries were obtained from local growers or from markets and were treated in a standard regime according to the methodologies used (spraying with chemical antifungal 4 weeks and 2 weeks before harvest). All samples were placed in separate cold boxes at 4 ° C during the first day after collection and fumigated and the first set of samples was collected. Subsequently, samples were taken after 4, 7, 11 and 18 days of storage and a visual assessment of the status of 1000 strawberries and a microbiological evaluation of the culture of 100 selected samples.

The results showed that both pre-harvest treatment and post-harvest fumigation had a significant effect on the level of fungal infection detected by culture microbiology. While in conventional control strawberries, fungi were found in 37% of analyzed strawberries after 4 days of storage, this value decreased to 14% after fumigation. However, if the strawberries were also pre-harvested by spraying, these values were 11% and 4% respectively.

The results in Table 2 then show a practically significant result of the assessment of the state of strawberries on a sample of 1000 fruits. When strawberries were sprayed with both pre-harvest and post-harvest biological agents, after 18 days of storage in the refrigerator at 4 ° C, only 3 percent of the decayed fruits were untreated, while the untreated control was essentially totally spoiled at that time

-6GB 32478 U1 rotting. In addition, a very interesting finding was found that the treated strawberries did not spread fungi from the affected to the treated fruits - so it was easy to pick up the spoiled fruits before sale and sell the rest of the intact fruits.

Table 2

Evaluation of the percentage of spoiled strawberries in the order of thousands evaluated under various conditions of post-harvest protection

Conditions 1 day (storage) (%) Day 4 (%) 7th day (%) 11th day (%) 18. den (%) 1. Inspection 5.2 11.6 20.7 on. on. 2. Control + Fumigation 1.4 3.5 11.0 on. on. 3. Treatment 0 0.3 1.9 4.9 9.8 4. Treatment + Fumigation 0 0.1 0.9 1,8 3.0

Table 2 shows the percentage of spoiled strawberries in terms of their storage time on days: 1, 4, 7, 11 and 18 days. The first column lists the 'conditions' for the treatment of strawberries:

'1. Control '- for strawberries, treated with chemical spraying only;

'2. Control + Fumigation '- for strawberries treated with post-harvest fumigation;

'3. Treatment '- for strawberries treated with a biological agent pre-harvest;

"4 Treatment + Fumigation" - for strawberries, pre-harvest and post-harvest treated.

The abbreviation “n.a.” means that it cannot be analyzed due to poor condition of the sample.

Example 3

Combined pre-harvest and post-harvest treatment of sour cherries

In cooperation with the orcharders based in the region of Biopreparáty spol. s ro in the Czech Republic experiments were carried out with combined pre-harvest and post-harvest protection of sour cherries using Polyversum. This preparation was applied in the pre-harvest regime at the rate of 100 g per 1 ha in an aqueous suspension with a sprayer, in post-harvest protection by fumigating the application biological agent in an amount of 150 g in 5 liters of an aqueous suspension per 500 m ^{3 of} storage area.

Pre-harvest application was carried out in accordance with patent EP 2503892 B1 of the owner Biopreparáty spol. s r.

In the post-harvest mode, treatment was then carried out with Pulsfog Bio K22 fumigators, with distilled water being used in the secondary cooling vessel and the biological composition of Table 1 in wt. %. In a specific case, 120 g of Polyversum biological fungicide were mixed with 200 g of food grade glycerol, 4676 g of distilled water and 4 g of calcium chloride dihydrate.

-7 GB 32478 U1

Control sour cherries were obtained from local growers or from markets (conventional chemical antifungal spraying 4 weeks and 2 weeks before harvest), were treated in a standard regime according to the above procedure. All samples were placed in separate refrigerator compartments at 10 ° C on the first day after collection, and samples were fumigated and a first set of samples was collected. Subsequently, samples were taken after 4, 7, 11 and 20 days of storage and a visual evaluation of the condition of the sour cherries and a microbiological evaluation of the selected samples were performed.

The results showed that both pre-harvest treatment and post-harvest fumigation had a significant effect on the level of fungal infection detected by culture microbiology: whereas in control sour cherries, fungi were found in 27% of analyzed sour cherries after 4 days of storage, this value decreased to 13%. However, if the cherries were also pre-harvested by spraying, these values were 10% and 4% respectively.

The results in Table 3 then show a practically significant result in a reduction in the incidence of Colletotrichos in sour cherries causing creasing and cracking of the fetus and discoloration. When sour cherries were sprayed with a biological composition according to the invention both pre-harvest and post-harvest, only 4 percent of the fruits were affected by Colletotrichose after 20 days of storage in the refrigerator at 10 ° C, while untreated controls were up to 35% at this time. In addition, a very interesting finding was obtained that the treated sour cherries did not spread from the affected to the treated fruit - so it was easy to pick up the spoiled fruit before sale and sell the rest of the intact fruit.

Table 3

Assessment of percentages of treated sour cherries, infested with Colletotrichos and / or Penicilium

Conditions Healthy (%) Colletotrichos (%) Penicilium (%) Colletotrichos + Penicilium 1. Inspection 29 35 13 23 2. Control + Fumigation 32 29 35 13 3. Treatment 31 9 55 7 4. Treatment + Fumigation 30 4 59 5

Table 3 lists the four 'conditions' for the treatment of sour cherries, namely:

'1. Control '- for sour cherries, treated only with a chemical spray;

'2. Controls + Fumigation '- for sour cherries treated after harvest fumigation;

'3. Treatment '- for sour cherries treated with a pre-harvest biological product;

„4 Treatment + Fumigation“ - for sour cherries, pre-harvest and post-harvest treated.

Table 3 relates in the following columns to the percentage evaluation of sour cherries infested with gray-green fungi Colletotrichos and / or Penicilium.

The description of the percentage of individual fungal diseases affected is as follows: 'Healthy': no disease was observed in sour cherries.

“Colletotrichos”: the presence of Colletotrichos has been observed in sour cherries.

'Penicilium': Penicilium has been observed in sour cherries.

“Colletotrichos + Penicilium in sour cherries has been observed to occur with Colletotrichos and Penicilium.

Example 4

Combined pre-harvest and post-harvest treatment of Chinese cabbage

In cooperation with Polish company BioAgris, business partner of Biopreparáty spol. s ro, experiments with combined pre-harvest and post-harvest cabbage protection using the biological fungicide Polyversum were carried out in fields in Poland and the Czech Republic. This preparation was applied in the pre-harvest regime in the amount of 100 g per 1 ha, in post-harvest protection by fumigation of the application biological agent according to the technical solution in the amount of 150 g in 5 liters of aqueous suspension per 500 m ^{3 of} storage area. Pre-harvest application was performed in accordance with the patent of the applicant Biopreparáty spol. s ro EP 2503892 B1; in the post-harvest mode, using Pulsfog Bio K22 or Pulsfog Bio K30 fumigators, with distilled water being used in the secondary cooling vessel and the biological composition of Table 1 used in the tertiary vessel of the present invention. In an embodiment, 200 g of Polyversum biological fungicide were mixed with 330 g of food or pharmaceutical grade glycerol, 4463 g of distilled water and 7 g of calcium chloride dihydrate.

Control cabbage was obtained from local growers or from markets (conventional spray with chemical antifungal 4 weeks and 2 weeks before harvest) and were treated in a standard regime according to the methodology used above. All samples were placed in separate cold boxes at 4 ° C during the first day after collection, and samples were fumigated and the first set of samples collected. Subsequently, samples were taken after 6 months of storage and a visual evaluation of the condition of 100 cones and a microbiological evaluation of the culture of 100 selected samples.

The results showed that both pre-harvest treatment and post-harvest fumigation had a significant effect on the level of fungal infection detected by culture microbiology: whereas in cabbage control heads, fungi were found in 57% of analyzed heads after 4 months of storage, this value decreased to 34%. If the cabbage was treated simultaneously with the pre-harvest spray, these values were 24% and 14% respectively. Cabbage growers also clearly showed that the affected cones in the treated variant were mold affected only at the cut point of the cone resulting from the harvest, so that the mildew affected area could be easily removed and the cone after sale for 4 months stored. Conversely, in the untreated crop variant, more than half of the cabbage heads were deeply affected by fungal infections, requiring either complete destruction of the affected head or significant mechanical trimming over the entire surface, accompanied by significant weight loss of the treated fetus.

Industrial applicability

The technical solution relates to the post-harvest protection of fruits and / or vegetables using new formulations of the microorganism Pythium oligandrum. The new solution can be used not only by individual gardeners and growers, but in combination with fumigation technology, the application is also suitable for incorporation into industrial post-harvest fruit and / or vegetable protection technologies in warehouses.

Claims (3)

What is claimed is: 1. A biological composition for post-harvest protection of fruit and / or vegetables, comprising: 2.0 to 4.0% by weight of a biological fungicide comprising 10 to 25% by weight of the milled substrate with the microorganism Pythium oligandrum M1, which has an oospore content of (1,0 ± 0,1) x 10 ⁶ per 1 g of biological fungicide and 75 to 90% by weight of silica; and further comprising a biological composition 3.34 to 6.60% glycerol; 0.06 to 0.14% by weight of calcium chloride dihydrate; and the remainder to 100 wt. is water. Biological composition according to claim 1 comprising an amount of the composition corresponding to a content of 100 to 200 g of biological fungicide as defined in claim 1 mixed with water to a volume of 5 liters for application per 500 m ^{3 of} fruit and / or vegetable storage space.