Method of internal seed disinfection by combining seed priming with vacuum infiltration

Abstract

Embodiments of the disclosed technology are directed to a method for combining seed priming and vacuum infiltration to facilitate deep penetration of chemicals or other substances into seeds. By combining the two processes, the germination process of seeds can be started allowing a relatively lengthy period of disinfection. The lengthy period of disinfection under vacuum provides maximum penetration into the seed in order to eradicate diseases occurring deep inside the seed. Any type of seed priming may be used in the disclosed method, including, but not limited to, osmopriming, matrix priming, or hydropriming. Further, any type of seed may be treated, including most vegetable crops, ornamentals, and agronomic crops.

Claims

1. A method of treating ungerminated seeds comprising: priming said seeds using a priming technique for a specific priming duration ranging from 1 to 15 days; and applying a vacuum infiltration technique for 6 hours or more to said seeds using a disinfecting solution, a total processing time, including seed priming and vacuum infiltration, is adjusted in order to bring seeds as close as possible to completing the germination process but stopping short of germination itself.

2. The method of claim 1, wherein said priming technique is osmopriming, matrix priming, or hydropriming.

3. The method of claim 1, wherein said priming technique is matrix priming.

4. The method of claim 1, wherein said priming technique is osmopriming.

5. The method of claim 1, wherein said disinfecting solution is electrolyzed water.

6. The method of claim 1, wherein said vacuuming duration is between 6 hours and 36 hours.

7. The method of claim 1, wherein said vacuuming duration is between 6 and 72 hours.

8. A method of treating ungerminated seeds comprising: choosing a treatment substance to be used in a disinfecting solution; performing a phytotoxicity screening of said treatment substance with respect to said seeds; priming said seeds using a priming technique for a specific duration ranging from 1 to 15 days; and applying a vacuum infiltration technique to said seeds using a disinfecting solution for at least 6 hours, a total processing time, including seed priming and vacuum infiltration, is adjusted in order to bring seeds as close as possible to completing the germination process but stopping short of germination itself.

9. The method of claim 8, wherein said phytotoxicity screening comprises: adding chemicals to water; and matrix priming said seeds using said water.

10. The method of claim 8, wherein said treatment substance is electrolyzed water.

11. The method of claim 8, wherein said treatment substance is an antibiotic.

12. The method of claim 8, wherein said treatment substance is a fungicide.

13. The method of claim 8, wherein said treatment substance is an anti-viral agent.

14. The method of claim 8, wherein said treatment substance is a biological agent.

15. The method of claim 8, wherein said seeds are vegetable crops.

16. The method of claim 8, wherein said seeds are agronomic crops.

17. The method of claim 8, wherein said seeds are ornamental crops.

18. A method of treating ungerminated seeds comprising: applying a vacuum infiltration technique for at least 6 hours to said seeds using a disinfecting solution; followed by priming said seeds using a priming technique for a specific priming duration ranging from 1 to 15 days, a total processing time, including seed priming and vacuum infiltration, is adjusted in order to bring seeds as close as possible to completing the germination process but stopping short of germination itself.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a flow chart outlining steps performed in an exemplary method of carrying out embodiments of the disclosed technology.

(2) FIG. 2 shows a table of the treatment results of an experiment involving oxytetracycline comparing methods of the prior art with methods of embodiments of the disclosed technology.

(3) FIG. 3 shows a table of the treatment results of an experiment involving different priming durations used in methods of carrying out embodiments of the disclosed technology.

(4) FIG. 4 shows a table of the treatment results of an experiment involving different priming durations and fluid amounts used in methods of carrying out embodiments of the disclosed technology.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE DISCLOSED TECHNOLOGY

(5) Embodiments of the disclosed technology are directed to a method for combining seed priming and vacuum infiltration to facilitate deep penetration of chemicals or other substances into seeds. By combining the two processes, the germination process of seeds can be started allowing a relatively lengthy period of disinfection. The lengthy period of disinfection under vacuum provides maximum penetration into the seed in order to exhume diseases occurring deep inside the seed.

(6) Any type of seed priming may be used in the disclosed method, including, but not limited to, osmopriming, matrix priming, or hydropriming. Further, any type of seed may be treated, including most vegetable crops, ornamentals, and agronomic crops. Virtually any substance that is soluble in water, such as fungicides, antibiotics, anti-viral agents, and biological agents can be used to treat seeds with this method providing they are not phytotoxic. In addition, water with biocidal properties such as electrolyzed water can be used as the aqueous solution in the vacuum infiltration process. When using matrix priming or hydropriming, both of which require an aqueous solution, water with biocidal properties such as electrolyzed water or plain water may be used as the aqueous solution. The combination of vacuum infiltration and seed priming can be carried out in any order, either vacuum infiltration first followed by seed priming, seed priming followed by vacuum infiltration, or vacuum infiltration in the middle of the seed priming process.

(7) Duration of the seed priming treatment varies by species. In embodiments of the disclosed technology, the duration of seed priming is decreased to allow time for vacuum infiltration. Total processing time, including seed priming and vacuum infiltration, is adjusted in order to bring seeds as close as possible to completing the germination process but stopping short of germination itself. Following the treatment, the seeds are dried and can be stored for later use. If treated properly, the seeds will be free of seed borne disease, exhibit higher seed vigor, have improved cold temperature germination, and possibly increased germination as well.

(8) Embodiments of the disclosed technology will become clearer in view of the following description of the figures.

(9) FIG. 1 shows a flow chart outlining steps performed in an exemplary method of carrying out embodiments of the disclosed technology. Step 110 involves choosing which species of seed will be sought to be disinfected. The next step, step 120, is directed to selecting appropriate substances that have been demonstrated to be effective against the target disease, and that are not phytotoxic. Since this is a new technique and penetrates deep in to the endosperm of seeds, new phytotoxicty screening needs to be done to see how a substance reacts under this method. Thus, in step 130, a phytotoxicty screening is carried out on the chosen seeds using the chosen treatment. Results of screening using prior methods are not applicable because other methods would have achieved lesser penetration and would not be an appropriate indicator of phytoxicity using the disclosed technology. For example, the inventors screened the antibiotic Oxytetracycline using the method of adding chemicals to the water used in matrix priming (Eastin, J. A., 2000, U.S. Pat. No. 6,076,301) and the method of the present invention. Water containing 500 ppm of Oxytetracycline was used to prime triploid watermelon seeds using matrix priming. Further, the same seed was treated with vacuum infiltration using 500 ppm Oxytetracycline solutions for 6 hours, which was followed by a matrix priming using water. For a control, seed of the same lot was treated using vacuum infiltration with water, followed by matrix priming using water. Also, untreated seeds were preserved as an additional control. The results are shown in the table 200 of FIG. 2.

(10) The results show that Oxytetracycline is highly phytotoxic when penetrating the seed endosperm. Germination was affected, and T50 (time in hours for 50% of seeds to germinate) was also affected. In addition, seedlings that developed from seeds treated with vacuum infiltration with a 500 ppm Oxytetracycline solution for 6 hours followed by matrix conditioning with water showed severe yellowing while others did not. Results demonstrate both the much deeper seed penetration when using the combination of seed priming and vacuum infiltration with chemicals as opposed to seed priming alone with chemicals, and also the phytotoxicity of Oxytetracycline. In this same manner, any substance can be checked for its suitability to this disinfection technique.

(11) Further screening using the above-described format, showed the following substances to be phytotoxic: Oxalinic acid, ciprofloxacin, chloramphenicol, and trisodium phosphate (a commonly used anti-viral substance). Substances screened showing mild phytotoxic effects include trimethoprim-sulfamethoxazole, colistin sulphate, Terraclor Super X (a commercial fungicide containing pentachloronitrobenzene and etridiazole), and Switch (a commercial fungicide containing fludioxonil and cyprodinil). Substances screened showing no phytotoxic effects are piperacillin/tazobactam, ceftriaxone sodium, and electrolyzed water.

(12) Substances with suitable levels of phytotoxicity are then screened for their affectivity against target diseases. For an example, the inventors screened the antibiotic piperacillin/tazobactam, and electrolyzed water for its affectivity against Acidovorax avenae subsp. citrulli on diploid watermelons seeds. Diploid watermelon seeds were chosen because they typically exhibit infections externally. Infected seeds were soaked in solutions for 30 minutes of electrolyzed water, and piperacillin/tazobactam at 100 ppm and 250 ppm. Results showed that electrolyzed water and piperacillin/tazobactam at 250 ppm were effective at eliminating the bacteria, while piperacillin/tazobactam at 100 ppm was not effective. This serves only as an example, and any method of screening can be used either in vivo or in vitro to screen for affectivity against target diseases and appropriate dose.

(13) Referring back to FIG. 1, if the screening produces unfavorable results, and the phytotoxicity is found in step 140, then the method is terminated in step 150. After step 150, the method may revert to step 120 whereby a new treatment substance may be chosen.

(14) If no phytotoxicty is found, then in step 160 a priming technique is chosen. Examples of priming techniques include osmopriming 161, matrix priming 162, and hydropriming 163. Proceeding with the method, in step 170, a priming duration is chosen. The priming duration is the length of time to which the seeds are subjected to a priming technique. The duration may range between a one day to a 15 days. The examples shown in FIG. 1 are two days 171, three days 172, and four days 173. Further, X days 174 is depicted to represent any duration prescribed by a user. Finally, in step 180, the seeds are treated using vacuum infiltration. For this step, a duration may be chosen as well, although the duration may not be as dispositive of results compared to the priming duration.

(15) Determination of proper length of priming treatment and vacuum filtration treatment are performed on an empirical basis. For an example, the inventors identified proper treatment length in the following manner. A lot of broccoli seeds infected with Xanthomonas campestris pv. Campestris were treated with the following treatment regiments. Osmotic priming with Polyethylene glycol (PEG) for 4, 5, and 6 days followed by vacuum infiltration with piperacillin/tazobactam at 500 ppm for 6 hours. Seeds were tested after treatments for both germination and presence of the disease Xanthomonas campestris pv. Campestris. The results are shown in the table 300 of FIG. 3.

(16) The results show that the best treatment for this particular lot was Osmotic priming for 4 days followed by 6 hours of vacuum infiltration with 500 ppm of piperacillin/tazobactam, which gave the best germination results and eradicated the seed borne disease Xanthomonas campestris pv. Campestris. This example shows the manner in which treatment protocols may be developed. Several standard protocols can be developed for each specific crop, and each lot is then tested to see which protocol is the best. As a starting point for protocol development, prior knowledge of the art of seed priming is used and then adaptation is made to suit the desired length of the vacuum infiltration process.

(17) As another example based on prior protocol development, melon seeds infected with Cucumber green mottle mosaic virus (CGMMV) were treated. First, the seeds were treated externally with trisodium phosphate to inactivate virus on the seed coast to ensure that infection is internal. Treatments were as follows: Matrix priming for 3 days with 100 grams seed, 300 grams solid matrix, 70 ml water followed by 24 hours vacuum infiltration with electrolyzed water, matrix priming for 3 days with 100 grams seed, 300 grams solid matrix, 100 ml water followed by 24 hours vacuum infiltration with electrolyzed water, matrix priming for 4 days with 100 grams seed, 300 grams solid matrix, 100 ml water followed by 24 hours vacuum infiltration with electrolyzed water, and matrix priming for 5 days with 100 grams seed, 300 grams solid matrix, 100 ml water followed by 24 hours vacuum infiltration with electrolyzed water. During the 24 hour vacuum infiltration period, the electrolyzed water was replaced with fresh electrolyzed water every hour. Seeds were tested for germination and for CGMMV by seedling ELISA. Results are shown in the table 400 of FIG. 4.

(18) According to the results, germination was improved, and time to 50% germination (T50) decreased by 42% showing superior speed in germination in cold temperature. Most importantly, seed borne disease was eradicated. As depicted in FIGS. 2 through 4, combining seed priming with vacuum infiltration before, during, or after priming, internal seed disinfection can be achieved while improving seed germination quality, and enabling storage for future use.

(19) The specific examples above are to be construed as merely illustrative, and not limitative of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. Although specific examples were given of seed borne diseases, the invention can be used for virtually all seed borne diseases, including bacteria, fungi, viruses, and viroids. In addition, specific crops were mentioned in the examples; however the invention can be used for seeds of virtually any crop including vegetables, agronomic crops, and ornamentals. The invention is an application technique, and can be used with any substance that is miscible in water.

(20) While the disclosed technology has been taught with specific reference to the above embodiments, a person having ordinary skill in the art will recognize that changes can be made in form and detail without departing from the spirit and the scope of the disclosed technology. The described embodiments are to be considered in all respects only as illustrative and not restrictive. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope. Combinations of any of the methods, systems, and devices described hereinabove are also contemplated and within the scope of the disclosed technology.