Method for disinfecting soils or other agricultural growing media

Abstract

The invention relates to a method for disinfecting soils or other agricultural growing media, characterised by comprising the following steps: obtaining a soil or other agricultural growing medium at their field capacity; treating the soil or medium at the field capacity of the previous step with ozonated water, wherein the ozonated water is prepared in situ with ozone-production equipment connected to the water supply; allowing a period of time to pass after the treatment with ozone; and inoculating the disinfected soil or agricultural medium with at least one species of beneficial microorganism.

Claims

1. A method for disinfecting soils or other agricultural growing media, comprising the following steps: obtaining a soil or other agricultural growing medium at their field capacity; treating the soil or medium at the field capacity of the previous step with ozonated water containing at least 2 ppm of ozone, wherein the ozonated water is prepared in situ with ozone-production equipment connected to the water supply with a pH between 5.6 and 6.5; allowing a minimum period of 30 min to pass after the treatment with ozone; and inoculating the disinfected soil or agricultural medium with at least one species of beneficial microorganism.

2. The method for disinfecting according to claim 1, wherein a period of between 30 min and 48 hours is allowed to pass between the treatment of the soil or medium with the ozonated water and the inoculation with at least one species of beneficial microorganism.

3. The method for disinfecting according to claim 1, wherein the content of ozone dissolved in the ozonated water is between 5 and 6 ppm.

4. The method for disinfecting according to claim 1, wherein the treatment with ozonated water is performed for a period of time between 1 h 30 min and 2 hours.

5. The method for disinfecting according to claim 1, wherein the content of ozone dissolved in the ozonated water is between 6 and 8 ppm, and the treatment of the soil or medium with this ozonated water is performed for a period of time less than 90 min.

6. The method for disinfecting according to claim 1, wherein the ozonated water prepared in situ contains nano-bubbles of ozone obtained in a mixing tank pressurised at a pressure between 2.5 and 3.0 atm.

7. The method for disinfecting according to claim 1, wherein the beneficial microorganism is selected from the group consisting of Trichodermas, Bacillus, Azotobacter, Pseudomonas and any one combination thereof.

8. The method for disinfecting according to claim 1, wherein the disinfected soil or agricultural medium is treated with 11 different strains of microorganisms.

9. The method for disinfecting according to claim 1, wherein the water is applied by localised irrigation.

10. The method for disinfecting according to claim 1, wherein the soil or medium does not have a crop in production, and the method comprises the following steps: preparing the soil or medium for planting; obtaining the soil or medium at their field capacity; treating the soil or medium at the field capacity of the previous step with ozonated water containing at least 2 ppm of ozone, wherein the ozonated water is prepared in situ with ozone-production equipment connected to the water supply with a pH between 5.6 and 6.5; allowing a minimum period of 30 min to pass after the treatment with ozone; inoculating the disinfected soil or agricultural medium with at least one species of beneficial microorganism; and planting the plant to be grown.

11. The method for disinfecting according to claim 1, wherein the soil or medium has a crop in production, and the method comprises the following steps: obtaining the soil or medium at their field capacity; treating the soil or medium of the previous step with ozonated water containing at least 2 ppm of ozone, wherein the ozonated water is prepared in situ with ozone-production equipment connected to the water supply with a pH between 15.6 and 6.5; allowing a minimum period of 30 min to pass after the treatment with ozone; and inoculating the disinfected soil or agricultural medium with at least one species of beneficial microorganism.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Diagram showing the effect of water with ozone on several fungi.

(2) FIG. 2a: Image showing the containers with medium used in example 1.

(3) FIG. 2b: Image showing the system used in example 1 in order to perform the disinfection of a medium according to the method of the present invention.

(4) FIG. 3a: Graph showing the survival of phytopathogenic fungi after application 1 of example 1. The amount of fungi present in the medium at time zero (t=0) and 24 hours after the application (t=1), at depths of 20 cm, 40 cm and 60 cm, is analysed.

(5) FIG. 3b: Graph showing the survival of phytopathogenic bacteria after application 1 of example 1. The amount of bacteria present in the medium at time zero (t=0) and 24 hours after the application (t=1), at depths of 20 cm, 40 cm and 60 cm, is analysed.

(6) FIG. 4a: Graph showing the survival of phytopathogenic fungi after application 2 of example 1. The amount of fungi present in the medium at time zero (t=0), 1 day (t=1) and 30 days (t=30) after the application, at depths of 20 cm, 40 cm and 60 cm, is analysed.

(7) FIG. 4b: Graph showing the survival of phytopathogenic bacteria after application 2 of example 1. The amount of fungi present in the medium at time zero (t=0), 1 day (t=1) and 30 days (t=30) after the application, at depths of 20 cm, 40 cm and 60 cm, is analysed.

(8) FIG. 5: Image showing the state of the tomato plants after the first application of example 1: FIG. 5a: control; FIG. 5b: treatment with ozone.

(9) FIG. 6: Image showing the state of the tomato plants after the second application of example 1. FIG. 6a: control; FIG. 6b: treatment with ozone.

(10) FIG. 7: Sampling points collected in example 3.

(11) FIG. 8: Images showing: (A) soil sample under a microscope; (B), (C) and (D) detail of nematodes.

(12) FIG. 9: Graph showing the evolution of the nematode population in the soil after the treatment with ozone and after the inoculation of microorganisms obtained in example 5.

EXAMPLES

Example 1: Effectiveness of the Treatment with Ozone in the Disinfection of Soils without Crops

(13) The general objective of the study was to evaluate the effectiveness of the use of ozone as a strategy in the disinfection of soil. In particular, the effect of the disinfection method on tomato plants was to be studied, as well as relating the oxidising power of the ozone with the elimination of pathogens at different depths.

(14) In order to achieve the aforementioned objectives, soil without crops was infected with the phytopathogenic species Sclerotinia, Fusarium, Erwinia, Phytophthora and Clavibacter. The assay was performed in Lorca (Murcia). It took place in open air in bins with soil without crops and wherein tomatoes, peppers and melons had been grown previously. After performing the first application of the method of the present invention, tomato plants, Lycopersicum esculentum, were transplanted, the response of the plants to the treatments being evaluated.

(15) The treatments tested were: T0 (control) and T1 (ozone). After the application of the method two consecutive times, the inoculation of the following beneficial microorganisms (mixture called “microorganism formula (1)”) was performed: Trichodermas (4×10.sup.8 UFC), Bacillus (1.5×10.sup.8 UFC), Azotobacter (1.5×10.sup.8 UFC) and Pseudomonas (1.5×10.sup.8 UFC).

(16) The microbiological content of the soil was evaluated at different depths: 20, 40 and 60 cm., before and after the applications.

(17) 1.1 Assay Protocol

(18) Containers were installed with a known volume of 1000 l (1×1×1 m) in field. Moreover, a medium proceeding from a greenhouse with melons, peppers and tomatoes having been grown there previously was selected and homogenised. Subsequently, the containers were filled with this medium.

(19) In the laboratory, the following species were cultivated in vitro: Phytopathogenic bacteria: Erwinia amylovora and Clavibacter michiganensis Phytopathogenic fungi: Sclerotinia sp, Fusarium oxysporum and Phytophthora citrophthora

(20) Each microorganism was grown in liquid medium until reaching a concentration of 10.sup.8 ufc/ml. For each of the bins, 1 litre of microbial solution was prepared.

(21) Then the pathogenic microorganisms were inoculated in the medium, periodic irrigations were performed until reaching the capacity of the medium, samples were taken at different depths (20 cm, 40 cm and 60 cm) and the phytopathogens were counted in said samples, in order to verify the permanence and presence of phytopathogens in the soil.

(22) A first application of the disinfection method with ozone was performed. To do so, the ozonation equipment was inserted into the irrigation system and the parameters of said equipment were adjusted in order to achieve ozonated water with at least 2 ppm of dissolved ozone. Subsequently, the soil was left to rest for 30 min and the tomato plants, Lycopersicum esculentum, were transplanted. Subsequently, a second application of the disinfection method with ozone was performed in the same conditions indicated previously and, after letting the treated soil rest for 30 min, the microorganism formula (1) was inoculated.

(23) FIG. 2a shows the placement of the containers in the field, while FIG. 2b shows the assembly made for inserting the ozonation equipment into the irrigation system.

(24) In order to analyse the effect of the treatment on the disinfection of the soil, samples were taken at different depths (20 cm, 40 cm and 60 cm) and on different dates, and the microbial load in each of the samples was analysed.

(25) Afterwards, as mentioned earlier, once the first disinfection was finished, tomato seedlings were transplanted into the bins in order to evaluate the “residual effect” of the treatment. Subsequently, the study of the plant development of the plants was performed.

(26) 1.2 Treatments

(27) The treatments, applications and sampling are referred to in the following table:

(28) TABLE-US-00001 Sampling Sampling Code Description Application 1 1.1 1.2 T0 Control 12-Sep 16-Sep T1 Ozone 15-Sep 12-Sep 16-Sep Sampling Sampling Sampling Code Description Application 2 2.1 2.2 2.3 T0 Control 14-Oct 17-Oct 14-Nov T1 Ozone 16-Oct 14-Oct 17-Oct 14-Nov

(29) After the first application of the disinfection method, on 19 Sep. 2014, 2 tomato plants were transplanted per container. The residual effect of the ozone was evaluated.

(30) 1.3 Experimental Size and Design

(31) The control treatment corresponds to container No. 1. Given that there is only one repetition, during the samplings, 3 subsamples were taken.

(32) TABLE-US-00002 TABLE 2 Outline of the experimental design Code Description Repetitions Container Code T0 Control 3 1, 2, 3 T1 Ozone (>2 ppm) 3 4, 5, 6
1.4 Parameters Evaluated

(33) Throughout the assay, the parameters evaluated were: Flow rate and ppm ozone at the outlet. Microbial evaluation of the soil (t=0; t=7 days and t=30 days). Relationship of the depth with the possible fungicidal effect. Having established the tomato plants, it was evaluated if the treatments caused phytotoxicity.

(34) The parameters that will be observed in the crop will be the appearance of necrosis and burning on leaves and fruits. Plant development of the crop.

(35) The effect of the treatments (ozone, control) and the evaluation thereof were determined by means of the Student test in the SPSS statistical packet. The test determines whether the measurements produced by the experiments are significantly different with a level of reliability (P>0.05). The results are shown by means of bar diagrams. In the results table, it is shown if there were significant differences (different letter) or if there is no evidence of differences between the treatments (same letter).

(36) 1.5. Results

(37) 1.5.1 Flow Rate and ppm Ozone at the Outlet

(38) During the first application, the data collected is shown in the following table:

(39) TABLE-US-00003 TABLE 3 Ozone readings Time Equipment (minutes) involved Description/Observations  0-120 Irrigation Water saturation 15-20 Ozone Verification on 4 occasions of the analysis in equipment order to determine the grams of ozone per litre Drip of water. 2 ppm. Minimum level for a good irrigation disinfection 15 Reading   2 ppm 55 Reading  2.3 ppm 65 Reading 2.89 ppm 115 Reading 4.49 ppm in the direct outlet of the pump 3.92 ppm in the dripper
1.5.2. Evaluation of the Microbial Population after Application 1

(40) In this section the data obtained is described. As explained in the assay protocol, soil samples were taken at different depths (20 cm, 40 cm and 60 cm) and quality controls were performed quantifying the number of pathogenic fungi and bacteria present.

(41) A. Pathogenic Fungi

(42) Prior to the incorporation of the treatments and 24 hours after the application, the survival of pathogenic fungi was evaluated. Having analysed the data by treatment (see FIG. 3a), we found that: In the control treatment: the microbial load was similar before and after at all the depths tested, except for 40 cm, wherein washing was produced. The ozone application caused a 100% reduction at depths of 20 and 60 cm.
B. Pathogenic Bacteria

(43) At each of the depths tested (20 cm, 40 cm and 60 cm), both treatments reduced the populations of bacteria present in the soil (see FIG. 3b). At all the depths tested (except for 60 cm), the populations of pathogenic bacteria were less than the control, this reduction being less than 50%.

(44) 1.5.3. Evaluation of the Microbial Population after Application 2

(45) A. Pathogenic Fungi

(46) As shown in FIG. 4a, the application of ozone reduced the pathogenic fungi by 100% at depths of 40 and 60 cm, maintaining the effectiveness of both products and prolonging the effect for 30 days at a depth of 20 cm, the treatment with ozone reduced the population by 100%, with traces of fungi appearing a month later, possibly due to contamination of the soil from environmental factors.

(47) B. Pathogenic Bacteria

(48) The study of the data (see FIG. 4b) shows the disinfecting power of the ozone, although the reduction was not complete. Given the reproduction speed of the bacteria, it is estimated that after the residual effect of the products disappear completely, populations similar to the control will be reached.

(49) 1.6. Phytoxicity in Tomato Plants

(50) After the first application of the treatment with ozone, 2 tomato plants were transplanted into each of the containers. The growth of the plants was evaluated for three months, ending the analysis of this parameter on 2 January due to frost in the area (see FIGS. 5a and 5b).

(51) The growth in plants wherein the treatment with ozone had been applied showed vigorous plant development, due to the decrease in pathogenic microorganisms; while in control plants, the development thereof was medium-low. Two weeks after the second application, the control plants died from the infection (see FIGS. 6a and 6b).

(52) 1.7. Conclusions

(53) The results obtained demonstrated that the disinfection level of the ozone was 100% against the phytopathogenic fungi tested. Additionally, the populations of pathogenic mesophilic bacteria decreased in number after the application of the disinfectant, with a mortality less than 50% after the first application. During the second application, the reduction of 50% was maintained, except for what was observed at a depth of 60 cm, wherein the reduction reached 60%.

(54) Furthermore, in the samples treated with ozone, the plant development of the tomato plants was vigorous due to the reduction of the populations of microorganisms, meanwhile, the tomato plants grown in control containers showed a low-medium growth and ended up dying due to the infection by pathogens.

(55) In addition, after the first application, tomato plants were transplanted into each of the repetitions and it was evaluated whether the application of ozone caused phytotoxicity. Three months after the transplant, plants treated with ozone exhibited normal growth and plant development, while the control plants died as a consequence of the attack of pathogens present in the soil.

Example 2: Disinfection of Nematodes in Padrón Peppers

(56) The assay was performed in a property with Padrón peppers in the month of May 2015 which is significantly affected by nematodes and the property intends to start planting since the production has already been considered lost. On a 500 m.sup.2 surface of a greenhouse, with a water consumption of 3000 l/h, the disinfection with ozone was performed at a dose of 6 ppm, revising with the REDOX readings that a reading of 1000 mV is obtained in the farthest drip emitters, starting from a value of 185 mV in the irrigation water without ozone.

(57) When the treatment ends, the inoculation of the microorganism formula (1) is performed (see example 1). Seven days after performing the treatment, samples of earth are taken to see the level of nematode infestation, observing a decrease of 90% of the population.

(58) In particular, after the disinfection through ozonated irrigation in a Padrón pepper crop affected by nematodes (Meloidogyne) in full production, the following results were obtained: Analytics before the disinfection: Meloidogyne sp. 88 juveniles/100 cc soil Analytics after the disinfection: Meloidogyne sp. 8 juveniles/1 cc soil.

(59) The treated portion of the property recovered from the damage and continued to produce until the date foreseen for the start thereof.

Example 3: Disinfection of Nematodes in Citrus Crops (Lemon Trees). Effect of Ozonation and Inoculation with Beneficial Microorganisms in the Control of Phytopathogenic Nematodes in Soil

(60) The present assay in citrus plants was performed in a parcel of land with a high index of nematode infection, during the months of May and September 2015. The effect of two ozone applications and a final application of a mixture of microorganisms (microorganism formula (2)) made of: Bacillus (1.5×10.sup.8 UFC), Azotobacter (1.5×10.sup.8 UFC) and Pseudomonas (1.5×10.sup.8 UFC) was evaluated.

(61) The parameters studied were: evolution of the phytopathogenic nematode population, evolution of the fungal population and after the application of microorganism formula (2), a count of the number of bacteria in the soil was performed. The results show that the application of the ozone together with beneficial microorganisms significantly reduced the population of phytopathogenic nematodes in the soil.

(62) An objective of this assay was to evaluate the action of ozone and beneficial microorganisms in the control of phytopathogenic nematodes in citrus plants.

(63) 3.1. Material and Methods

(64) On a parcel of land with a high infection by nematodes of the genus Pratylenchus sp, 4 repetitions were selected. FIG. 7 shows the sampling points gathered.

(65) The application date of the products was: Ozone: applied at a concentration of 5-6 ppm. Applications: 21 and 27 May. Microorganism formula (2) (Bionema Plus): A single application of a dose of 20 l/ha took place on 4 July.

(66) TABLE-US-00004 TABLE 1 Samplings Sampling Description of the application Date Sampling 1 Before application 20-May Sampling 2 After application 1 of Ozone 21-May Sampling 3 After application 2 of Ozone 28-May Sampling 4 After application of Bionema Plus 23-Sep
3.2. Conclusions

(67) The results have shown that the application of the ozone and the microorganism formula (2) significantly reduced the population of phytopathogenic nematodes, decreasing from 50 nem/g to 0.2 nem/g, the population being practically non-existent.

Example 4: Evolution of Nematodes in the Soil after Treatment with Ozone and Inoculation of Microorganisms

(68) This assay was performed in T. M. Benifaió (Valencia), in clay-loam terrain. The treatments were performed at the end of a crop of vegetables, before performing the new planting of vegetables such as cucumbers, tomatoes, peppers.

(69) The terrain was notable for the high content of nematode infection thereof, severely affecting the production of the crop.

(70) The method for disinfecting soils which is described in this patent application was applied, wherein the treatment with ozonated irrigation water (5.5 ppm of ozone) was performed for 2 hours, and the inoculation took place by means of the application of the colonising microorganism formula (1) (see example 1) 24 hours after performing the disinfection with ozone.

(71) As can be observed in FIG. 9, before applying the treatment the soil sample taken had 480 juveniles/100 cc of earth. After performing the treatment with ozone, the presence of juveniles was reduced to 44/100 cc of earth and 15 days after performing the inoculation with the microorganism formula (1), the sample analysed had 1 juvenile/100 cc of earth.

(72) These results show a considerable decrease in the percentage of nematodes. Furthermore, an increase can also be seen in the amount of saprophytes in 20 units/100 cc of earth.

(73) The different assays performed show that, both in bare terrain as well as terrain with crops in production, the best results are obtained by combining the punctual treatment with ozone (at least 2 ppm) in irrigation water, followed by the inoculation with beneficial microorganisms.