Bacterial formulation for biocontrol of plant diseases and promotion of plant growth

Abstract

The present invention relates to bacterial formulation in vegetable fiber materials, which can be employed by incorporation into soils and by treating seeds, as well as in nutrient in hydroponic systems, for the promotion of plant growth and/or the biologic control of diseases. Preferably, the formulation of the present invention employs coconut fiber. An advantage of the present invention is the possible replacement of turf and other organic or inorganic materials employed in the formulation of inoculants and of agents for the biologic control of bacteria and fungi by coconut fiber.

Claims

1. A package comprising a formulation comprising effective amounts of: a) Pseudomonas chlororaphis and/or Pseudomonas aureofaciens; b) coconut fiber; c) water, in an amount such that said formulation has a moisture content of at least 45%; d) optionally, nutrient additives or growth factors selected from the group consisting of sugars, amino acids, proteins, salts and seeds, or mixtures thereof; and e) optionally, adjuvants selected from the group consisting of osmotic regulatory agents, buffering agents and pH adjusting agents.

2. The package according to claim 1, wherein said formulation further comprises a microorganism from a family, order or phylum selected from the group consisting of Actinobacteria, Aquificae, Bacteroidetes, Chlorobi, Chlamydiae, Verrucomicrobia, Chloroflexi, Chrisiogenetes, Cyanobacteria, Gloeobacteria, Nostocales, Oscillatoriales, Pleurocapsales, Prochlorales, Stigonematales, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Fibrobacteres, Acidobacteria, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteriz, Spirochaetes, Thermodesulfobacteria, and Thermotogae.

3. The package according to claim 1, wherein said formulation is formulated for the biological control of diseases and pests.

4. The package according to claim 3, wherein said pests are selected from the group consisting of caterpillars, cankerworms, acaridae, clothes moths, worms, beetles, cochineal insect, white flies, grass-hoppers, flea-insects, cochonilhas (Coccus viridis), centipedes, plant lice, spiders, ants, and insect larvae.

5. The package according to claim 3, wherein said diseases are caused by bacteria, fungi, zoopore producing pathogens, pathogens carried to the soil, root pathogens and pathogens of aerial parts of plants.

6. The package according to claim 5, wherein said pathogens carried to the soil are selected from a genus in the group consisting of Pythium, Phytophthora, Fusarium, and Rhizoctonia.

7. The package according to claim 1, wherein said formulation is able to promote plant growth.

8. The package according to claim 1, wherein said formulation is in a form selected from the group consisting of: a composition for suspension in water; a composition suspended in water; a dispersible formulation; grains; and a material for coating seeds and other propagation organs.

9. A method for the biological control of plant pathogens, comprising administering an effective amount of the formulation of the package according to claim 1 to seeds, seedlings, or a propagation material intended for plantation.

10. A method for the biological control of plant pathogens, comprising administering an effective amount of the formulation of the package according to claim 1 to a plant in need thereof in a culture substrate and/or in a nutrient solution.

11. A method for the biological control of plant pathogens, comprising administering an effective amount of the formulation of the package according to claim 1 to hydroponic cultures, substrates for producing seedlings, substrates for growing plants, or to soils.

12. A method for controlling a biological pest or disease, comprising administering an effective amount of the formulation of the package according to claim 1 to a plant in need thereof, or to seeds, seedlings, or a propagation material intended for plantation.

13. A method for promoting plant development, comprising administering an effective amount of the formulation of the package according to claim 1 to a plant in need thereof, or to seeds, seedlings, or a propagation material intended for plantation.

14. The package of claim 1, wherein said formulation contains between 110.sup.7 to 110.sup.11 colony forming units (cfu)/mL of said Pseudomonas chlororaphis and/or said Pseudomonas aureofaciens.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows graphs illustrating the survival (shelf-life) of Pseudomonas chlororaphis 63-28 in coconut fiber, with or without addition of carboxymethylcellulose (CMC) or xanthan gum (XAN) at temperatures of 31 C. (A) and 221 C. (B).

(2) FIG. 2 shows graphs illustrating the survival (shelf-life) of Pseudomonas aureofaciens TX-1 (A-B) and Pseudomonas chlororaphis 63-28 (C-D) and in coconut fiber with different moistures at temperatures of 31 C. and 221 C.

(3) FIG. 3 shows graphs illustrating the survival of Pseudomonas chlororaphis 63-28 in water, canola oil and buffer MgSO4 0.1 M at temperatures of 31 C. (A) and 221 C. (B).

(4) FIG. 4 is a graph showing foliar growth of a young leaf of hydroponic lettuce plant after infestation or without infestation of the nutrient solution with Pseudomonas chlororaphis 63-28 cells, multiplied by two days, or Pseudomonas chlororaphis 63-28, formulated in coconut fiber and stored for 140 days.

(5) FIG. 5 is a graph showing the foliar growth rate of hydroponic lettuce plants after infestation or without infestation of the nutrient solution with Pseudomonas chlororaphis 63-28 (2 days) or Pseudomonas chlororaphis 63-28 (140 days).

DETAILED DESCRIPTION OF THE INVENTION

(6) The present invention relates to a bacterial formulation in vegetable fiber materials and may be used by incorporating the formulation into soils and in treating seeds, as well as in nutrient solutions in hydroponic systems, to promote plant growth and/or biological control of diseases.

(7) The formulation of the present invention is characterized by comprising: (a) microorganism cells; (b) vegetable fiber material; (c) water in an amount sufficient to keep the microorganism cells feasible; (d) optionally, nutrient additives or growth factors; (e) optionally, adjuvants selected from osmotic regulating agents, buffering agents, pH adjusting agents.

(8) The term microorganism refers to microscopic organisms such as bacteria, virus, fungi and protozoa. Preferably, the present invention has, as microorganisms, the organisms chose from the group of bacteria. Bacteria refer to prokaryotic organisms, with the exception of cyanophyciae. The invention has use on various species of bacteria, which include, but are not limited to the group of Actinobacteria, Aquificae, Bacteroidetes/group Chlorobi, Chlamydiae/group Verrucomicrobia, Chloroflexi, Chrisiogenetes, Cyanobacteria, Gloeobacteria, Nostocales, Oscillatoriales, Pleurocapsales, Prochlorales, Stigonematales, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Fibrobacteres/group Acidobacteria, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteriz, Spirochaetes, Thermodesulfobacteria, Thermotogae. Preferably, the invention makes use of bacteria selected from the group of plant-growth promoting rhizobacteria, including Pseudomonas spp. Herbaspirillum spp., Azospirillum spp., Gluconacetobacter spp., Burkholderia spp., and Bacillus spp. More preferably, the invention relates to Pseudomonas.

(9) The microorganism cells used in the present invention are defined as being colony forming units. Preferably, for the present invention one has used from 110.sup.7 to 110.sup.11 colony forming units (cfu)/mL.

(10) As used herein, the expression vegetable material means each and every material from vegetables such as barks/husks and other fibrous components from seeds and fruits, plant stems. Preferably, for the present invention one has used coconut fibers. The coconut fiber of the present invention can be used as follows: ground in different grain sizes, associated with additives for acidity correction electric conductivity. The amount of coconut fibers used will depend on the grain size thereof, and may range from 1 mm to 10 mm. This grain size will depend on the use of the formulation. For hydroponics, seed treatment, spraying and irrigation, it will be as small as possible (<2 mm). On the other hand, for mixture with substrate or soil, it may be from 1 to 10 mm. Preferably, for the present invention the coconut fiber has been sieved in 2-mm opening sieve and used in the ratio 10:1 (10 g of coconut fiber: 1 ml of bacterium cell suspension.

(11) The present invention is characterized in that the nutrient additives or growth factors are selected from the group consisting of sugars, amino acids, proteins, salts and the like, or mixture thereof.

(12) The present invention further works with adjuvants selected from osmotic regulating agents, buffering agents or pH adjusting agents. Preferably, the present invention uses, as adjuvants, carboxymethylcellulose, gum Arabic, sodium alginate and the like. Preferably, the invention uses calcium carbonate (CaCO.sub.3) as a pH adjusting agent to neutralize it in the formulation.

(13) The formulation of the present invention can be used in the biological control of diseases and pests. The pests on which the formulation act may be, but are not limited to, pests of the group containing caterpillars, cankerworms, acaridae, clothes moths, worms, fungal insects, beetles, cochineal insect, white flies, grass-hoppers, flea-insects, cochineal insect, centipedes, plant lice, spiders, ants, and insect larvae. The diseases on which the formulation acts may be caused by fungi, bacteria, zoospore-producing pathogens, pathogens carried into the soil, root pathogens and pathogens of the aerial part of plants. The control of disease of the aerial part of plants is made by means of the resistance inducing mechanism. Particularly, the formulation of the present invention acts on organisms of the kingdom Stramenopilae of the phylum Oomicota of the family Pythiaceae of the genus Pythium, and zoospera-producing pathogens.

(14) The formulation of the invention can be applied to seeds and any propagating material intended for plantation. The formulation of the invention may be suspended in water or dispersible, powder, granules or in the form of a seed coating powder.

(15) The formulation of the invention can be applied in hydroponic cultures, seedling producing substrates, substrate for plant growth, soils, nutrient solutions and irrigation water. In nutrient solution and irrigation water, the formulation can be applied directly in tanks. On the other hand, in substrates and soils, it may be mixed before the seeding/planting and afterward via irrigation water.

(16) The invention also describes a method characterized in that one applies the formulation of the invention in an amount effective to control a biological past. The expression effective amount may be defined as being the amount necessary to kill or reduce a determined crop pest, or the amount necessary to enable the development of plants. The amount of formulation will depended on the type of pest and crop. For the present invention, one used, as a biological model, the pathosystem lettucePythium aphanidermatum. Preferably, for the present invention the final concentration employed was of 10.sup.7 ufc/mL of nutrient solution.

(17) The formulation of the invention can be applied to seeds, seedlings and any propagating material intended for plantation. The propagating materials of the present invention may be, but are not limited to seedlings, seeds, stalk, bubbles, branches, stock for grafting, stem and all the plant parts liable to be propagated.

(18) The formulation of the invention can further be applied to cultivating substrate and in the nutrient solution.

(19) The invention also describes a method characterized in that one applies the formulation in an amount effective for enabling the development of hydroponic plants, as well as a seedling producing substrate, plant growth substrate and soils. The amount of formulation to enable the development of a plant will depend on the type of crop. For the present invention, one has studied the cultivation of lettuce. The formulation was applied in the nutrient solution, by adding suspensions in the final concentration of 10.sup.7 ufc/mL of nutrient solution. The formulation for use in hydroponic crops should have adequate grain size (smaller than 2 mm), so that the system will not be clogged. Formulations for use in hydroponic crops that use organic substrate do not have restrictions as to the grain size.

(20) The formulation of the present invention can be used in the form of water suspension or dispersible, in granules, or in the form of a powder for coating seeds and other propagation organs.

EXAMPLES

(21) The present invention is further defined in the following examples. One should understand that, although these examples indicate part of the invention, they are given with a view to illustrate the invention, without being limitative of the scope of the present inventions.

Example 1Obtainment of the Bacteria

(22) The bacterial isolates sued in the present invention were Pseudomonas aureofaciens, strain Tx-1, and Pseudomonas chlororaphis, strain 63-28. Both isolates belong to the crop collection of the Guelph University in Canada and were obtained from the company Eco Soils Systems, Inc., San Diego, Canada.

Example 2Multiplication of the Bacteria

(23) Pseudomonas spp. Isolates were cultured in a liquid medium Tryptic Soy Broth (TSB3-10 g/liters of water) per 48 hours in an agitator at 150 rpm at the temperature of 221 C. After 48 h, the bacterial suspension was centrifuged at 2000 g for 15 min, the cells being re-suspended and washed by centrifugation at 2000 g for 10 min in buffer MgSO.sub.4 0.1 M.

Example 3Application of the Bacterial Cells in Coconut Fiber

(24) For evaluation of the shelf-life of Pseudomonas spp., in coconut fiber, it was necessary to sieve the fiber. For this purpose, it was ground and sieved with a 425 m opening sieve, and to neutralize the pH (7) with CaCO.sub.3.

(25) In polypropylene sacks having openings for oxygen exchange, one has added the volume of 90 mL of ground and sieved coconut fiber, previously sterilized, the sterilization being carried out by autoclaving for three alternating days at 120 C. the bacterial suspensions at a concentration of 5109 colony forming units (ufc)/mL were added to the coconut fiber, so that the coconut fiber moisture could be adjusted to 75-80%. The polypropylene sacks containing the coconut fiber with the bacterial suspensions were added at 31 C. and 221 C., so that the temperature of 31 C. could provide longer shelf-life.

(26) Weekly or monthly evaluations were carried out to assess the bacterial population by plating in a TSB medium plus agar.

Example 4Evaluation of the Shelf-Life of Pseudomonas chlororaphis 63-28 and Pseudomonas aureofaciens TX-1 in Coconut Fiber with Different Units

(27) In order to evaluate the shelf-life of P. chlororaphis 63-28 and P. aureofaciens TX-1 in coconut fiber (Optimum Hydroponix, Canada), one sieved the fiber with 425-m-opening and neutralized the pH with CaCO3. The volume of 90 mL of coconut fiber was added in polypropylene sacks having openings for oxygen exchange. The sterilization of the substrate was carried out by autoclaving for 3 alternating days at 120 C. After the autoclaving, the bacterial suspensions were added to the coconut fiber. The moistures coconut fibers were adjusted to 25%, 45% and 75%. The polypropylene sacks containing the coconut fiber with the bacterial suspensions were packed at 31 C. and 221 C. for 120 days. Monthly evaluations were carried out to assess the bacterial population by plating in TSB medium plus agar, and the moisture of the substrates.

Example 5Evaluation of the Shelf-Life of Pseudomonas chlororaphis 63-28 in Coconut Fiber, Talc, and Turf

(28) Longer shelf-life of P. chlororaphis 63-28 in coconut fiber, either with or without addition of the additives carboxymethylcellulose or xanthan gum, was verified at 31 C., when compared with the shelf-life of the formulations stored at 221 C. (FIG. 1).

(29) Statistic differences were found in the 19.sup.th, 32th, and 37.sup.th storage weeks, the survival of the bacterium being higher in coconut fiber without additives at 31 C. (FIG. 1A). The addition of carboximethylcellulose did not increase the survival of the bacteria, and the addition of xanthan gum had a negative effect on the survival of P. chlororaphis 63-28 at 31 C. (FIG. 1A).

(30) When stored at 221 C., the bacterial population dropped by one log ufc mL.sup.1 unit of substrate after three weeks. No difference was found with regard to the additives to the formulation, keeping the same log ufc mL.sup.1 unit in the coconut fiber formulations with or without additives until five storage weeks (FIG. 1B). After 9-week storage at 221 C., the smaller bacterial population was found in the coconut fiber with addition of xanthan gum, and no statistical differences were found between the coconut fiber with or without carboxymethylcellulose (FIG. 1B). Statistic differences were also found after 72-week storage, and a larger bacterial population in coconut fiber with xanthan gum was found, followed by coconut fiber without additives, and with greater survival in coconut fibers with addition of carboxymethylcellulose (FIG. 1B).

(31) The bacterial formulations in turf and talc did not provide the maintenance of the survival of the bacterium in large populations, and they dropped by three log ufc mL.sup.1 units after application thereof to substrates (data not shown).

Example 6Evaluation of the Shelf-Life of Pseudomonas chlororaphis 63-28 and Pseudomonas aureofaciens TX-1 in Coconut Fiber with Different Moistures

(32) The temperature of 31 C., FIG. 2) was more efficient in keeping the shelf-life of Pseudomonas spp., when compared with the temperature of 221 C. (FIG. 2). The Pseudomonas spp formulation in coconut fiber with moisture of 75% provided greater bacterial survival when compared with 45% or 25% moisture (FIG. 2). The moisture of 25% caused a greater reduction in the bacterial population along the time, regardless of the bacterial species employed (FIG. 2).

(33) The population of P. aureofaciens TX-1 at 31 C. had a similar behaviour in coconut fiber with moistures of 45% and 75% until 60 days' storage. On the 90.sup.th day of storage, the bacterial population was higher in the substrate with 75% moisture and did not differ on the 120.sup.th day of storage. Coconut fiber with 25% moisture exhibited the lowest values of bacterial population ion all the periods evaluated (FIG. 2A).

(34) When stored at 221 C., the population of P. aureofaciens TX-1 was higher in coconut fiber with 75% moisture on the 30.sup.th day of storage, followed by coconut fiber with 45% and 25% moisture, respectively (FIG. 2B). On the 60.sup.th day of storage no differences were found between the moistures 75% and 45%, the moisture 25% providing the lowest survival. On the 90.sup.th and 140.sup.th days of storage, the coconut fiber with 75% moisture provided better survival of P. aureofaciens TX-1, when compared with the moisture of 45%. It was not possible to recover the bacterial cells in the culture medium of the coconut fiber with 25% moisture on the 90.sup.th and 140.sup.th days of storage (FIG. 2B).

(35) The P. chlororaphis 63-28 formulation ion coconut fiber with moisture of 75% at a temperature of 31 C. remained at the same log ufc mL.sup.1 unit of substrate during the 140 days of storage (FIG. 2C). On the 30.sup.th day, no statistic differences were found in the bacterial population between the moistures of 75% and 45%. However, the moisture of 25% exhibited the lowest value of log ufc (FIG. 2C). During the 60 and 90 days of storage, the bacterial population was higher at moisture 75%, followed by 45% and 25%, respectively. On the 120.sup.th day, one found greater bacterial population in the coconut fiber with 75%, and no differences were found at 45% and 25% at a temperature of 31 C. (FIG. 2C).

(36) The storage of P. chlororaphis 63-28 at 221 C. caused reduction of one log ufc mL.sup.1 unit of substrate in the bacterial population at moisture of 75% on the 90.sup.th day of storage (FIG. 2D). On the 30.sup.th day, the population of P. chlororaphis 63-28 exhibited the highest values of log ufc when the moisture of 75%, followed by 45% and 25%, respectively (FIG. 2D). During the 60 to 120 days of storage, the bacterial population was similar in the coconut fiber with 75% or 45% of moisture, with the same values of log ufc in the substrate with 25% moisture (FIG. 2D).

Example 7Evaluation of the Shelf-Life of Pseudomonas chlororaphis 63-28 in Water, Canola Oil and Magnesium Sulphate Buffer

(37) The temperature of 31 C. provided better survival of P. chlororaphis 63-28 than the temperature of 221 C., regardless of the suspension employed for preserving the bacterium (FIG. 3). With regard to the suspensions employed, the use of water and buffer provided better bacterial survival when compared with the canola oil (FIG. 3). The survival of P. chlororaphis 63-28 at the temperature of 31 C. was higher in water after 15 and 120 days' storage, followed by buffer and canola oil, respectively (FIG. 3A). During 30, 60 and 90 days' storage, no statistic differences were found between water and buffer. However, canola oil exhibited the lowest values of log ufc when its survival was considered (FIG. 3A). Statistic differences were not found with regard to the bacterial population in water and buffer throughout the experiment at 221 C. (FIG. 3B). However, the canola-oil suspensions exhibited the lowest values of ufc in all the periods evaluated (FIG. 3B).

Example 8Promotion of the Growth of Hydroponic Lettuce by Pseudomonas chlororaphis 63-28

(38) From the fifth days after infestation of the nutrient solution with bacterial suspensions, one found greater foliar growth of the plants in the treatments with infestation of the bacterial suspensions (FIG. 4).

(39) The date in FIG. 5 show higher foliar growth rate in the lettuce plants infested with bacterial isolates. Folia growth picks were found on the 2.sup.nd and 5.sup.th days after infestation of the nutrient solution with P. chlororaphis 63-28 (formulated in coconut fiber and with 140 days' storage) or without infestation with the bacterial cells, and on the 2.sup.nd, 4.sup.th and 6.sup.th days after infestation of the nutrient solution with P. chlororaphis 63-28 (multiplied by 2 days and used without formulation) (FIG. 5). However, the values of the areas below the growth curves and the foliar growth rates were not significant by the test F (Table 1).

(40) TABLE-US-00001 TABLE 1 Effect of infestation of the nutrient solution, or no infestation, of hydroponic lettuce with Pseudomonas chlororaphis 63-28, multiplied by two days or Pseulomonas chlororaphis 63-28 formulated in coconut fiber and stored for 140 days, on the 8.sup.th day after infestation of the plants on the foliar growth area and the area of foliar growth rate. Treatments Areas of foliar growth Area of growth rate Witness 938.27* 64.76 Pseudomonas chlororaphis 976.73 71.82 (2 days) Psudomonas chlororaphis 967.95 72.21 (140 days) *Data without letter were not significant by the F test.

(41) Infestation of the nutrient solution with P. chlororaphis preserved in coconut fiber for 140 days promoted the development of the aerial system of hydroponic lettuce, statistically differing from the witness without inoculation (Table 2). The employ of bacterial cells cultured in TSB for two days promoted the development of the aerial system of the plants, but the difference was not statistically significant with respect to the witness treatment without infestation with the bacteria (Table 2). The mass data of the root system did not differ by the F test (Table 2).

(42) TABLE-US-00002 TABLE 2 Effect of infestation, or absence of infestation, of the nutrient solution of hydroponic pimiento with Pseudomonas chlororaphis 63-28 cultured for two days or formulated in coconut fiber and preserved at 3 1 C. for 140 days on the mass of hydroponic lettuce. Fresh, Dry, of Fresh, of of the Dry, of the the aerial root the aerial root Dray, system system Fresh, system system total Treatment (g) (g) total (g) (g) (g) Witness 88.65*b 8.33* 96.98 b 5.02 b 0.36 5.38 b Pseudomonas 92.76 b 8.47 101.23 b 5.27 ab 0.38 5.65 ab chlororaphis (2 days) Psudomonas 103.13 a 8.69 111.82 a 5.79 a 0.39 6.18 a chlororaphis (140 days) *data followed by the same letter were not significant in the LSD test, at 5%. **Data without letter were no significant in the F test.