SEED TREATMENT

Abstract

The invention provides a seed treatment method comprising: (i) mixing starting materials (7) comprising an aqueous liquid (2), anaerobic bacteria (1), a substrate (3) for the anaerobic bacteria, and seeds (4), to provide a mixture (10) having a void volume (15) between the seeds, wherein at least 50% of the void volume comprises the aqueous liquid, (ii) storing the mixture in a condition to prevent oxygen from entering the mixture during a treatment time, to provide treated seeds (9), wherein the treatment time is at least 6 hours and at maximum 14 days, and (iii) collecting the treated seeds (9) from the mixture (10).

Claims

1. A seed treatment method comprising: (i) mixing starting materials comprising an aqueous liquid, anaerobic bacteria, a substrate for the anaerobic bacteria, and seeds, to provide a mixture having a void volume between the seeds, wherein at least 50% of the void volume comprises the aqueous liquid, (ii) storing the mixture in a condition to prevent oxygen from entering the mixture during a treatment time, to provide treated seeds, wherein the treatment time is at least 6 hours and at maximum 14 days, and (iii) collecting the treated seeds from the mixture.

2. The method according to claim 1, wherein the seeds are submerged in the aqueous liquid and wherein the aqueous liquid is water.

3. The method according to claim 1, wherein the pH of the mixture is selected from the range of 4-9.

4. The method according to claim 1, wherein the treatment time is selected from the range of 12-48 hours.

5. The method according to claim 1, wherein at least a part of the anaerobic bacteria in the mixture is provided by an anaerobic inoculum.

6. The method according claim 1, wherein the mixture at least during the treatment time is contained in a container to prevent oxygen from entering the mixture.

7. The method according to claim 6, further comprising evacuating the container and maintaining the container at a pressure below 1 bar during at least part of the treatment time.

8. The method according to claim 1, wherein the substrate for the anaerobic bacteria comprises at least one carbon source selected from the group comprising of a protein, a carbohydrate, and a lipid.

9. The method according to claim 1, wherein the anaerobic bacteria comprise anaerobic bacteria of the phylum of Firmicutes.

10. The method according to claim 1 wherein the minimum dimension of the seedsis selected in the range of 0.1-10 mm.

11. The method according to claim 1, wherein the seeds are selected from the group consisting of edible seeds, agricultural seeds, and horticulture seeds.

12. The method according to claim 1, wherein the seeds are selected from the group consisting of pasture crop seeds, cereals, legume seeds, and horticulture crop seeds.

13. The method according to claim 1, wherein the method is used for reduction of pathogens at the seeds.

14. The method according to claim 1, wherein the method is used to improve the germination of the seeds.

15. The method according to claim 1, wherein the seeds have a ratio of bacteria to pathogens equal to or larger than at least 10.sup.5: 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

[0081] FIG. 1 schematically depicts an embodiment of the method;

[0082] FIG. 2 schematically depicts two embodiments and some further aspects of the seed treatment method;

[0083] FIG. 3 schematically depicts another embodiment of the method; and

[0084] FIGS. 4a-c schematically depict some embodiments of ways to prevent oxygen to enter the mixture;

[0085] FIGS. 5a-b schematically depict some further aspects of the method.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0086] In FIG. 1 schematically the stages of some embodiments of the seed treatment method are depicted: in a first stage (A) a mixture 10 is made comprising the seeds 4 to be treated with an aqueous liquid 2, anaerobic bacteria 1, a substrate 3 for the anaerobic bacteria and optionally a solid material 5. In stage B the active anaerobic environment may be established by having microorganisms in the mixture being fed by the substrate while consuming the oxygen and preventing new oxygen to enter the mixture 10. In the anaerobic environment the anaerobic metabolism cleans the seed. In this stage, stage C, the anaerobic bacteria may feed themselves by the substrate and corpses of aerobic micro-organism killed by anaerobic environment, and the population of anaerobic microorganisms grows. After the anaerobic stage, the treated seeds 9 are separated from the mixture 10 and collected in stage D. The collected and treated seeds may still be covered with a small amount of substrate that when stored in air may feed (a small amount of) aerobic microorganism still present at the seeds and a new and more diversified microorganism population may grow. Hence the method also promotes an antagonistic process after killing of the pathogens. The schematically depicted stages in embodiments may be different in other embodiments. Some embodiment, e.g. comprise a pre-mixing or settling stage (see also below), wherein not all of the components of the starting materials may be introduced in the first stage (A) and wherein initially an anaerobic environment may be established before the seeds are added. In embodiments the bacteria may be provided by one of the other components in the mixture. In yet other embodiments, the bacteria may for instance be provided by an anaerobic inoculum (see also below).

[0087] In FIG. 2 schematically an embodiment of the method of the invention is depicted. In the figure at the top side an embodiment comprising a mixture 10 comprising a liquid matrix 11 is depicted, wherein the starting materials 7 comprise an aqueous liquid 2, anaerobic bacteria 1, a substrate 3 for the anaerobic bacteria 1, and especially the amount of aqueous liquid 2 being at least that much that the seeds 4 will be in the mixture 10 and are covered with aqueous liquid (see also FIGS. 5a-5b). The starting materials 7 are mixed with the seeds 4 to provide a mixture 10. Successively the mixture 10 is stored in a condition to prevent oxygen from entering the mixture; in FIG. 2 schematically depicted by having a lid 112 or any other oxygen barrier 110 hermetically closing the vessel 100 and therewith forming a closed container 130 to prevent oxygen from entering the mixture 10. After a specific treatment time, especially for the liquid matrix being in the range of 12-48 hours, the treated seeds 9 are provided and may be collected from the mixture 10, for instance by a sieve 140 separating the treated seeds 9 from the aqueous liquid 2. Further, the pH of the mixture 10 may be selected in the range of 4-9, especially in the range of 5-8.

[0088] At the bottom side of the figure an embodiment comprising a mixture 10 comprising a solid matrix 12 is schematically depicted. In this embodiment the starting materials 7 further comprise a solid material 5, and especially the amount of solid material 5 (in relation to the amount of the aqueous liquid 2 and the seeds 4 is selected to provide a moist (not submerged in the aqueous liquid) mixture 10. In embodiments, the mixture 10, especially comprising solid material 5, comprises an amount of seeds 4 selected in the range of at least 1 wt. % especially at least 5 wt. %. Even more especially at least 10 wt. %, such as 15-50 wt. % of the mixture 10 contains seeds 4. The solid material 5, especially for instance soil 6 that is not dried before use, may already comprise some water or aqueous liquid 2. The solid material 5 may comprise different kinds of solid, especially particulate material, such as sand or soil 6. Especially using soil 6 may be advantageous since the soil 6 may already comprise the anaerobic bacteria 1, and no extra anaerobic bacteria 1 such as in the form of an anaerobic inoculum 1 (see top figure with liquid matrix 11) has to be provided. Next to the solid material 5 also aqueous liquid 2 is required in the mixture 10. However the amount of aqueous liquid is much less and preferably the weight of the aqueous liquid 2 is about 10-35% of the weight of the solid material 5. Especially for the solid matrix 12 the treatment time is preferably in range of 3-14 days. Optionally, for both the solid and the liquid matrix 10, the container 130 may be evacuated, for instance by a vacuum pump 200, and a pressure below 1 bar may be maintained during at least part of the treatment time to decrease the oxygen content in the mixture 10. Also the used substrate 3 may affect how fast the oxygen concentration decreases in the mixture. Preferably, the substrate 3 for the anaerobic bacteria 1 is selected to provide for at least 75% of the treatment time an oxygen concentration 25 of less than 3% in the gaseous phase in the mixture 10.

[0089] In FIG. 3 an extra settling step is schematically depicted. The stage being introduced to lower the amount of oxygen in the mixture 10 before adding the seeds 4 and to increase the amount of anaerobic bacteria 1 in the mixture 10/in the solid matrix 12. In this embodiment the solid material 5, the substrate 3 for the anaerobic bacteria, the anaerobic bacteria 1 (being part of the solid material 5 and/or optionally added anaerobic bacteria 1) and at least a part of the amount of aqueous liquid 2 are mixed to provide a pre-mixture 20. The pre-mixture 20, next is stored during a settling time in a condition to prevent oxygen from entering the pre-mixture 20, at least until the oxygen concentration 25 in the gaseous phase of the pre-mixture 20 is less than 3%. The settling time, especially is in the range of 7-14 days. In the depicted embodiment, the pre-mixture 20 is stored in a closed container 130 to prevent oxygen to enter. After the settling time, the rest of the amount of the aqueous liquid 2 is mixed with the pre-mixture 20 to provide the mixture 10 and the seeds 4 may be mixed in the mixture 10 (like depicted in FIG. 2).

[0090] FIG. 4 schematically depicts some possible embodiments of an oxygen barrier (layer) 110 to prevent oxygen to enter the mixture 10 during the treatment. In FIG. 4a schematically an embodiment is given wherein the mixture 10 is laid on a surface, for instance the ground 190. A plastic film 111 is configured to be the oxygen barrier 110 and applied by covering the mixture 10. This embodiment may advantageously be applied with a solid matrix 12, wherein for instance the mixture comprises a local soil 6; the seeds 4 may be mixed with the soil 6, an aqueous liquid 2 and a substrate 3 and optionally extra anaerobic bacteria 1 and may be laid on the ground 190 and successively be covered by a barrier 110 to establish the anaerobic environment. In other embodiments the plastic film 111 or other barriers 110 may also be positioned at the surface 190 before laying down the mixture 10, so the barrier 110 may substantially enclose the mixture. In FIG. 4b an embodiment is depicted comprising a vessel 100 with a lid 112, wherein the mixture 10 is enclosed. Especially, such an embodiment may be applied for substantially liquid 11 as well as substantially solid 12 mixtures 10. Yet another embodiment is depicted in FIG. 4c. In this embodiment, the mixture 10 is substantially enclosed in an enclosure 113 configured as and oxygen barrier 110. At the top of the enclosure 113 an opening 114 is depicted used to fill the enclosure. This opening 114 is shown for clarity only and may be closed during the treatment time. In FIG. 4 only a few embodiments are depicted exemplifying the method. Many other embodiments are feasible to prevent oxygen to enter the mixture 10 as will be understood by a person skilled in the art. Embodiments may for instance comprise a silo, a container, a place with walls with a concrete floor. For quantities of several tons of seed move-able concrete separation walls of fixed concrete walls may be used to keep one batch separated from another batch. The concrete may be covered with a liner, large enough to cover the walls to prevent oxygen entering the (pre-)mixture. Yet other embodiments may comprise a (grain) silo or ship (a cargo hold).

[0091] In FIG. 5a-b schematically some embodiments of the mixture 10 are depicted, especially in relation to the method. In FIG. 5a a liquid matrix 11 is schematically depicted comprising the seeds 4 and with a void volume 15 between the seeds 4. In the depicted embodiment substantially 100% of the void volume 15 is comprised by the aqueous liquid 2. Especially the seeds 4 are submerged in the aqueous liquid 2. The mixture 10, further comprises anaerobic bacteria 1 and a substrate 3 in the aqueous liquid 2 (and thus in the void volume 15). The aqueous liquid 2 especially comprises water. Hence, the entire void volume 15 is filled with aqueous liquid 2 comprising anaerobic bacteria 1 and a substrate 3.

[0092] In embodiments at least 50% of the void volume 15 comprises the aqueous liquid 2. The remainder of the void volume 15 not comprised by the aqueous liquid 2 may comprise for instance bacteria, substrate, gas (pockets), solid material. Especially the remainder of the void volume 15 may comprise solid material 5, especially one or more of soil 6 and sand. In the embodiment schematically depicted in FIG. 5b the void volume 15 further comprises solid material 5. Hence the void volume 15 comprises the aqueous liquid 2, solid material 5, bacteria 1, and substrate 3. Especially in such embodiment, less than 50% of the void volume 15 may comprise aqueous liquid 2. Especially in such embodiment also at least 50% of the void volume 15 may comprise aqueous liquid 2. Especially, however in such embodiment comprising a solid matrix 12, less than 50% of the void volume 15 may comprised aqueous liquid 2 and more than 50% of the void volume 15 may comprise solid material 5. In the solid matrix 12, as schematically depicted in FIG. 5b, the seeds 4 may also not have a high packing density (due to the presence of other solids).

REFERENCE

[0093] 1 anaerobic bacteria [0094] 2 aqueous liquid [0095] 3 substrate [0096] 4 seeds [0097] 5 solid material [0098] 6 soil [0099] 7 starting materials [0100] 9 treated seeds [0101] 10 mixture [0102] 11 liquid matrix [0103] 12 solid matrix [0104] 15 void volume [0105] 20 pre-mixture [0106] 25 oxygen concentration [0107] 100 vessel [0108] 110 oxygen barrier [0109] 111 plastic film [0110] 112 lid/closure [0111] 113 enclosure [0112] 114 opening [0113] 130 container [0114] 140 separator [0115] 190 ground [0116] 200 vacuum pump

EXAMPLES

Effect Different Treatments of the Invention

[0117] The effects of different techniques of seed treatments on different seeds were studied experimentally. Herein the seeds (Spinach, Cauliflower, Brachiaria, and Carrot (two types) have been treated in a liquid matrix and a solid matrix. The air has been evacuated from both matrices after mixing the mixtures, and the techniques are respectively indicates by liquid priming and vacuum bags. In the vacuum-bags, seeds are vacuum packed together with a substrate (comprising protamylase) having a 20% protein content and soil (intrinsically already comprising anaerobic bacteria), wherein about 2.5 grams of protein is added per liter of soil. In the liquid priming experiments, seeds are suspended in a solution of water, the same substrate (2.5 grams of protein per liter of water) and an anaerobic inoculum. The treatment time is varied (<5, 28 and 60 hours) as well as the pH's. Germination improved through liquid-priming except for 60 hours with most extreme pHs. Just as with vacuum-bags, in-house microbiological assay suggested a change in microbial diversity with liquid-priming, especially with treatments of 28 and 60 hours. The germination of the treated seeds have been compared to untreated seeds as well as to federal standards (minimum required percentage that germinate) Also the morphology change because of the treatment is evaluated.

[0118] Some of the results are further summarized in the next table. It is concluded that there was a large improvement in germination after certain treatments. Most of the germinations after the treatments were higher than the minimum percentage germination according to federal standards, with the minimum germination to federal standards: spinach=60%, cauliflower=75%, carrot=55%, see Handbook for vegetable growers, 1988, J. E. Wiley&Sons, Inc.; Vegetable growing handbook 1979. Splittstoesser, W. E. AVI Publishing, Inc.; Seeds, the yearbook of Agriculture 1961, Stefferud, A., Editor. The United States Government Printing Office. No physically abnormal germination, disorder and discolor of seeds were observed. Apparently no phytotoxicity was observed in any treatment. The reduction in germination after some treatments showed that the treatment effect highly depends on the types of seeds. It also is concluded that small seed types (cauliflower and carrot) do not well reacted to the vacuum bag treatments, whereas this method works better for big seeds with hard seed coat (spinach and Brachiaria). It was also concluded that the optimal treatment time for different types of seeds was different. For Brachiaria seeds, there is no data available for the germination; however research shows that normally new harvest, matured, and undamaged Brachiaria seeds are highly dormant. During the experiment, the tested seed lot showed a germination % around 40% for untreated seeds.

[0119] No 100% elimination was obtained by the disinfection, however certain treatments decreased the appearance of fungi during the germination, especially vacuum bag treatment for spinach and Brachiaria seed did. Since cauliflower and carrot seeds were not heavily infested by fungi, only minor disinfection effects are shown after the treatments.

TABLE-US-00001 TABLE The effects of the seed treatment Treat- Seed Treatment Germination Germination ment Type time increase I increase II: Morphology L S 43 hours +20% +32% 42%>3% S S 3 days +38% +52% 42%>25% S S 6 days +33% +47% 42%>30% L C 43 hours 0% +23% 5%>1% S C 3 days +3% +27% 5%>25% S C 6 days 5% +16% 5%>6% L B 43 hours +60% NA 51%>53% S B 3 days +60% NA 51%>32% S B 6 days +85% NA 51%>35% L CA 43 hours +6% +59% 2%>0% S CA 3 days 4% +44% 2%>1% S CA 6 days 16% +27% 2%>0% L CB 43 hours +5% +52% 1%>0% S CB 3 days 2% +43% 1%>1% S CB 6 days 36% 7% 1%>0%

[0120] In the Table following abbreviations are used, Treatment: L=liquid priming; S=Vacuum bags; Seed: S=Spinach; C=Cauliflower; B=Brachiaria; CA=Carrot, type A; CB=Carrot, type B. Explanation of the columns: Germination increase I=germination of treated seed compared to germination of un-treated seeds, wherein the germination of the controls were spinach=66%, cauliflower=92%, Brachiaria=20%, carrot A=83%, carrot B=80%

[0121] Germination increase II=germination increase of treated seeds compared to minimum germination given by federal standards: spinach=60%, cauliflower=75%, carrot=55%. NA=no data available.

[0122] Change in morphology indicating the % of fungi infested seeds before & after treatment.

Visual Microbiological Counting

[0123] The development of fungi in treated and non-treated seeds after using the seed treatment method was compared. Microorganism morphology was noted in a presence/absence matrix (1/0 table). The categories for which the controls showed the same morphology were isolated. Patterns were found in five categories microorganisms. The treatment changed occurrence of micro-organism in comparison to control. This is also the case for two categories in the treatment with sand instead of soil. Three main expressions of microorganisms are (spontaneously) induced (Opaque wool+Tips, White Wool, Grey Silver spots). This phenomenon is most pronounced at highest moisture levels and longest treatment time (300 hours). Microorganisms are also eradicated (Black spots on seeds, Yellow-Orange veins on blotter paper).

[0124] The experiment shows that there is a switch in the typology of fungi expressed before and after the treatment. Non-treated infected seeds express black and grey spots. Treated seeds tend to show white molds, like Opaque wool+Tips, White Wool.

TABLE-US-00002 TABLE The effects of the seed treatment on presence/absence of categories Veins on Fuzz on seeds paper Opaque wool + White Spots on seeds Yellow- Treatment Tips wool Silver Black Orange Untreated 0 0 0 1 1 Dry soil 0 0 1 1 0 Soil 6 days treat 0 1 1 0 0 Soil 3 days treat 0 1 1 0 0
microorganism (Opaque wool+tips and White wool observed as Fuzz on seeds; Grey Silver and Black observed as spots on seeds; and Yellow-Orange Veins (fungi) on paper). Explanation of the columns: presence=1; absence=0.

Treatment of Brachiaria Brizanta Tropical Grass Seed.

[0125] A pre-mixture is prepared (or re-used) by mixing liquid substrate with sieved soil in a concrete mixer. In each concrete mixing batch about 20 kg of soil is mixed with liquid substrate to reach a protein content of 3%. Water is added to make the soil moist but below water capacity. No free water is visible after the mixing. If there is too much water, some dry soil is added.

[0126] The pre-mixture soil is dumped in a heap on a concrete floor covered with a plastic liner that blocks oxygen well. A second piece of the same type of liner is used to cover the heap and sand bags are used to keep the top liner fixed to the floor. The concrete floor is in the shade, covered by a roof and in a well ventilated area with walls with large openings or no walls to some sides. After 1 week of fermentation the pre-mixture soil can be used for the next step in the process.

[0127] Fermented pre-mixture soil of 1 week old is mixed with dry seeds inside a concrete mixer. A calculated amount of water is added to keep the mixture at soil capacity. If a water activity meter is present then the aim is to obtain a water activity of 0.985 or slightly higher. The ratio of seed : pre-mixture is 1:6.

[0128] This mixture is again dumped on a concrete floor covered with an oxygen blocking plastic liner. After shaping and compressing the heap a second liner is put over the heap. Sand bags are used to keep the liner in place and fixed on the floor. The mixture is then kept for 6 days under the closed conditions. After the treatment the seeds and the soil is separated by a screen to collect the treated seeds and proceed to preparing them for commercial purposes .

[0129] Survivability of treated seeds increased between 10 and 20% compared to the control.