Formulation comprising a beneficial <i>P. bilaii </i>strain and talc for use in seed treatment

Abstract

The present invention relates to a method of treating seed, comprising applying at least one plant treatment agent to said seed; and applying a dry formulation comprising/consisting of spores of P. bilaii and talc powder to said seed. The invention furthermore relates to a formulation for use in seed treatment comprising spores of P. bilaii and talc powder, seed treated with such formulation and methods of improving applicability and quality of P. bilaii spores and methods of improving plant growth, plant health or plant vigor.

Claims

1. A method of treating seed, comprising a) applying a liquid comprising at least one seed treatment agent to said seed and b) applying a dry formulation comprising spores of the fungus Penicillium bilaii and talc powder to the seeds, wherein the at least one seed treatment agent is selected from the group consisting of a plant protection agent, a plant growth promoting agent, a seed coating agent or an inoculant.

2. The method of claim 1 wherein step a) and b) are performed simultaneously.

3. The method of claim 1, wherein the seed is selected from the group consisting of Canola, oilseed rape, wheat, soybean, cotton, sugar beet, beans, corn, alfalfa, peas, lentils, flax and barley seed.

4. The method according to claim 1, wherein the seed is Canola seed.

5. The method of claim 1, wherein said plant protection or plant growth promoting agent is a chemical agent.

6. The method of claim 1, wherein said plant protection agent is a biological control agent.

7. The method of claim 1, wherein said plant protection agent comprises a chemical agent selected from the group consisting of azoxystrobin, benzovindiflupyr, boscalid, cyprodinil, fludioxonil, fluxapyroxad, iprodione, metalaxyl, mefenoxam, metconazole, penthiopyrad, picoxystrobin, propiconazole, prothioconazole, pyraclostrobin, sedaxane, tebuconazole, penflufen, clothianidin, trifloxystrobin, cyantraniliprole, fluquiconazole, fluopyram, fluoxastrobin, flupyradifurone, thiamethoxam, chlorantraniliprole, sulfloxaflor, difenoconazole, picarbutrazox, ethaboxam, chlorantraniliprole, chlorpyrifos, deltamethrin, dimethoate, imidacloprid, cyazypyr, cyhalothrin-lambda, permethrin, spirotetramate, and tetraniliprole and a combination thereof and/or a biological agent selected from the group consisting of Bacillus subtilis strain QST713, Bacillus subtilis strain GB03, Bacillus firmus strain I-1582, Bacillus pumilus strain QST 2808, Bacillus pumilus strain GB34, and Bacillus subtilis strain MB600, and a combination thereof.

8. The method of claim 1, wherein said plant protection agent is in a liquid formulation.

9. The method of claim 1, wherein in step b) said seed is treated with a formulation consisting of spores of P. bilaii and talc powder.

10. A method according to claim 1 wherein the Penicillium bilaii spores and talc powder are provided in a ratio of between 1:100 and 1:10.

11. A method of improving plant growth, plant health or plant vigor comprising growing seed obtained by the method of claim 1.

12. A seed treated according to the method of claim 1.

13. A method of improving plant growth, plant health or plant vigor comprising growing the seed of claim 12.

Description

(1) The present invention is illustrated by the following examples in a non-limiting fashion.

Example 1: Production of a Talc-Based Formulation of P. Bilaii

(2) Spores of P. bilaii strain deposited under DSMZ 32334 were obtained from solid-state fermentation and separation from the fermentation substrate. 40 g of spores were mixed with 960 g of talc powder to obtain the talc-based formulation.

Example 2: Seed Treatment with the Formulation According to the Invention

(3) Canola seed previously treated with a liquid composition comprising penflufen, clothianidin, metalaxyl, trifloxystrobin (commercialized as Prosper Evergol, Bayer CropScience), were treated with the formulation according to Example 1 when they were still sufficiently humid to make the formulation stick to the seeds.

(4) Application rates range from 2.5 gr to 12.5 grams /kg of seed to result in a concentration of viable fungal spores of at least 1 to 5×10.sup.9 spores per kg.

Example 3: Viability of P. Bilaii in the Dry Formulation Comprising Spores of P. Bilaii and Talc Powder

(5) Viability of P. bilaii spores is determined by measuring the amount of colony forming units (cfu). The method of the determination of the number of viable spores is based on the determination of colony-forming-units (cfu) per gram of sample. The sample is suspended in sterile Aqua dest. and diluted to suitable dilution steps. Subsamples of the dilution steps are plated on petri dishes with a solid potato-dextrose-agar. The petri dishes are incubated at 25° C. for 5-7 days until fungal colonies appear. According to the number of counted colonies and the dilution steps the cfu per gram sample is calculated.

(6) The spore content of a formulation obtained according to the protocol of Example 1 was measured and was at least 4×10.sup.9 viable spores/ g. while Jumpstart comprises 7.2×10.sup.8 per gram of formulated product. Samples of the formulation were stored at 4, 20 and 30° C. for 6 months and the viability of the spores tested once per month. Viability of the fungal spores at 4° C. and 20° C. did not significantly decrease after 6 months.

Example 4: Heubach Dust Off Testing for 2 Canola Seedlots with Different Thousand Seed Weight (tsw)

(7) The seeds were treated according to the invention with different amounts of a formulation according to the invention (comprising 4 w.-% P. bilaii spores, corresponding to 4.53×10.sup.9 cfu/g) and different concentrations of the commercially available product Jumpstart (1.63×10.sup.9 cfu/g), also comprising talc as drying agent, each followed by coating step with mica 300 g/100 kg. Treatment was done in cold temperature.

(8) The methodology is based on the approved European Seed Association (ESA) test method for analyzing dust generation.

(9) Treated seeds were mechanically stressed inside a rotating drum. A vacuum pump creates an air flow through the rotating drum, the connected glass cylinder and the attached filter unit. By the air flow, abraded dust particles are transported out of the rotating drum through the glass cylinder and subsequently through the filter unit. Coarse non-floating particles are separated and collected in the glass cylinder while floating dust particles are deposited onto a filter. The amount of floating dust collected on the filter is determined gravimetrically.

(10) A starting sample must consist of at least 500 g +/−5% of seeds. A subsample (100±1 grams) of treated seed was weighed and transferred into the metal drum of the Heubach device. The drum was then correctly closed, reassembled, and connected to the glass cylinder. A glass fiber filter disc (Whatman GF 92 or similar specification) was placed in the filter unit. The filter unit including the filter disc was weighed, placed on the glass cylinder and connected to the vacuum tube. The “time” option (2 min) was selected from the control panel and the rotation cycle started. After completion of the run, the filter unit including the filter disc was removed from the glass cylinder and weighed in the same manner as described before. The test was conducted on 3 subsamples. After each measurement, all components which are in contact with seed or dust (ie. rotating drum, glass cylinder, filter unit) were cleaned using a vacuum cleaner with a pointed nozzle.

(11) The Heubach dust value is expressed in g/100 kg of treated seeds. The following formula is used to convert the measured result to the Heubach dust value:

(12) $\mathrm{Heubach}\mathrm{dust}\mathrm{value}=\frac{\left(W_1-W_0\right)\mathrm{\aleph 100}\mathrm{.000}}{W_s}\left[g\text{/}100\mathrm{kg}\right]$
where: W1=weight of the loaded filter unit incl. filter disc [g] W0=weight of the empty filter unit incl. filter disc [g] WS=weight of the treated seeds [g]

(13) TABLE-US-00001 Calculated grams of dust/100 kg Trt Variety 1 Variety 2 # Treatment description tsw ~6.5 g tsw ~3.5 g 1 Prosper Evergol + talc Current commercial application 1.1924 0.6324 (500 g/100 kg) 2 Prosper Evergol + [(P. bilaii Replace talc with P. bilaii 0.4397 0.3428 spores + talc) spores + talc, low rate (250 g/100 kg)] 3 Prosper Evergol + [(P. bilaii talc replaced with high application 0.3799 0.3032 spores + talc) rate of P. bilaii (1250 g/100 kg)] spores + talc 4 Prosper Evergol + Jumpstart Low rate comparison - delivers 1.2795 0.6496 63 g slurry + talc similar number of spores JS* vs (500 g/100 kg) dry formulation 5 Prosper Evergol + Jumpstart High rate comparison - delivers 4.0917 1.1627 315 g slurry + talc similar number of spores JS vs (500 g/100 kg) + dry formulation mica (300 g/100 k) 6 Prosper Evergol + Jumpstart Current commercial application 1.4683 0.4659 80 g slurry + talc (500 g/100 kg) + mica (300 g/100 k) *JS = Jumpstart

(14) Treatments 2 and 4 as well as 3 and 5 comprise comparable amounts of P. bilaii spores, respectively. Treatment 6 corresponds to the recommended application rate.

(15) According to the table shown above, treatment of seeds with the dry formulation as described herein results in less dust generation due to abrasion of seed treatment material, in particular when high spore amounts are used. As seed treatment in Canada may be performed on seeds having a temperature below 0 degree C., the use of less liquid, in particular aequous liquid in the formulation of the present invention will increase the efficiency and feasibility of the treatment process.

Example 5: Germination Testing for 2 Canola Seedlots

(16) The seeds treated in Example 4 were assessed for germination rates.

(17) The methodology is based on the approved International Seed Testing Assocation (ISTA) test methods.

(18) Individual 10 cm×10 cm plastic germination trays with friction sealing lids were prepared by cleaning and then placing an appropriately sized blue blotter into each tray. 50 seeds were taken at random from each treatment sample and spaced uniformly and adequately apart on top of the moistened substrate using a vacuum counter. 4 trays were set up for each treatment for a total of 200 seeds per treatment. Trays were incubated in a closed cabinet set at 20 degrees C. and a 12 hour day/night light/dark cycle. Germination evaluations for canola were made 7 days after planting and according to ISTA guidelines. To be assessed as a normal seedling the seedling must have all their essential structures well developed, complete, in proportion and healthy.

(19) TABLE-US-00002 Trt # Treatment Description Variety 1 Variety 2 Average 1 Prosper Evergol + talc 82.5 92.5 87.5 (500 g/100 kg) 2 Prosper Evergol + [(P. bilaii 78 94.5 86.25 spores + talc) (250 g/100 kg)] 3 Prosper Evergol + [(P. bilaii 81.5 93 87.25 spores + talc) (1250 g/100 kg)] 4 Prosper Evergol + Jumpstart 84 92 88 63 g slurry + talc (500 g/100 kg) 5 Prosper Evergol + Jumpstart 80.5 90.5 85.5 315 g slurry + talc (500 g/100 kg) 6 Prosper Evergol + Jumpstart 82 89 85.5 80 g slurry + talc (500 g/100 kg) 7 Untreated 88 88 88

(20) Treatments 2 and 4 as well as 3 and 5 comprise comparable amounts of P. bilaii spores, respectively. Treatment 6 corresponds to the recommended application rate.

(21) The table above shows comparable germination rates for both the commercially available Jumpstart formulation and the dry formulation comprising spores of P. bilaii and talc powder.

Example 6: Viability of Penicillium Bilaii Spores After Application to Canola Seed

(22) Evaluation of on-seed viability of Penicillium bilaii on hybrid canola seed.

(23) Hybrid canola seed was treated in a Hege 11 seed dresser with seed treatment formulations followed by mica (300 g/100 kg) and the treated seed stored at room temperature ˜20° C. for 1 day.

(24) To extract the spores from the seed surfaces, 5 g of seed was weighed and added to a 100 mL flask containing 45 mL of steril water +0.1% Tween. Flasks were put on a shaker platform for 30 mins at 200 rpm. 2 replicates were set up for each treatment.

(25) A dilution block was prepared with 900 μL phosphate buffered saline (PBS) solution +0.1% Tween. The dilution series was carried out starting with 1mL of solution from the extraction flask to 1.00E-03. 100 μL from each dilution was then plated on PDA+Streptomycin Sulphate+Chloramphenicol+Triton X-100 plates; 2 replicates were plated for each sample (6 plates/sample). Plates were incubated in the dark at 20° C. for 5 days. Emerging colonies were marked, counted, and incubated further for confirmation. Calculations were then made to determine the number of colony forming units (CFUs) per gram of seed.

(26) TABLE-US-00003 Hybrid 1 Hybrid 2 AVERAGE 1 Prosper Evergol + [(P. bilaii 1.88E+06 3.04E+05 1.09E+06 spores + talc) (250 g/100 kg)] 2 Prosper Evergol + [(P. bilaii 2.13E+05 1.09E+05 1.61E+05 spores + talc) (1250 g/100 kg)] 3 Prosper Evergol + Jumpstart 1.63E+05 2.58E+04 9.41E+04 63 g slurry + talc (500 g/100 kg) 4 Prosper Evergol + Jumpstart 3.55E+05 7.43E+04 2.15E+05 315 g slurry + talc (500 g/100 kg) 5 Prosper Evergol + Jumpstart 1.18E+05 2.99E+04 7.38E+04 80 g slurry + talc (500 g/100 kg)

(27) Treatments 1 and 3 as well as 2 and 4 comprise comparable amounts of P. bilaii spores, respectively. Treatment 5 corresponds to the recommended application rate.

(28) This experiment shows that viability of the spores is improved compared to the viability of the experiments using Jumpstart.