Coating compositions for pathogen control in monocotyledonous plants

Abstract

Coating composition for applying to a plant structure of a monocotyledonous plant from which roots and shoots are capable of growing, wherein the said coating composition comprises an organic carrier material and one or more biological agents that possess an activity against at least one or more pathogens of the said monocotyledonous plant.

Claims

1. A coating composition in dry powder form for application to the surface of a monocotyledonous plant structure or a seed of a monocotyledonous plant, the coating composition consisting essentially of: (i) particles consisting of at least one organic carrier material and having a volume mean diameter of 5 μm to 200 μm, wherein the carrier material is selected from waxes having a melting point of ≥54° centigrade, and (ii) one or more biological agents that possess an activity against at least one pathogen of a monocotyledonous plant: wherein the particles of the at least one organic carrier material are thermally bonded to said one or more biological agents.

2. The coating composition according to claim 1, wherein the volume mean diameter of the particles is in the range of 8 to 200 μm.

3. The coating composition according to claim 1, wherein the biological agent is selected from a chemical agent and a live biological agent or is a mixture thereof.

4. The coating composition according to claim 1, wherein the biological agent is selected from chemical fungicides, arthropodicides and bactericides or is a mixture of two or more thereof.

5. The coating composition according to claim 4, wherein the arthropodicides are insecticides or acaricides.

6. The coating composition according to claim 1, wherein the at least one pathogen is a bacterial species, a fungal species or an arthropod species.

7. The coating composition according to claim 1, wherein the biological agent is at least one biological antagonist that is present in the form of bacterial spores and/or fungal spores located on the surface of the said particles.

8. A method of manufacturing the dry monocotyledonous seed coating composition according to claim 1 that comprises: 1) selecting an organic carrier material from waxes having a melting point of ≥50° Centigrade; 2) comminuting said organic carrier material into particles of a volume mean diameter ≥5 μm; and 3) adding to the particles produced in step 2) one or more biological agents that possess an activity against at least one pathogen of a monocotyledonous plant.

9. A seed product of a monocotyledonous plant comprising: (1) a seed of a monocotyledonous plant; and (2) the coating composition as defined in claim 1 in dry powder form on the surface of the seed.

10. A coating composition in dry powder form for application to the surface of a monocotyledonous plant structure or a seed of a monocotyledonous plant, the coating composition consisting essentially of: (i) particles consisting of at least one organic carrier material and having a volume mean diameter of 5 μm to 200 μm, wherein the carrier material is selected from waxes having a melting point of ≥50° centigrade; and (ii) one or more biological agents that possess an activity against at e one pathogen of a monocotyledonous plant; wherein the particles are made by a process comprising the steps of: (1) providing the organic carrier material; (2) comminuting said organic carrier material into particles of a volume mean diameter ≥5 μm; and (3) adding to the particles produced in step (2) the one or more biological agents.

11. The coating composition according to claim 10, wherein the volume mean diameter of the particles is in the range of 8 to 200 μm.

12. The coating composition according to claim 10, wherein the biological agent is selected from a chemical agent and a live biological agent or is a mixture thereof.

13. The coating composition according to claim 10, wherein the biological agent is selected from chemical fungicides, arthropodicides and bactericides or is a mixture of two or more thereof.

14. The coating composition according to claim 13, wherein the arthropodicides are insecticides or acaricides.

15. The coating composition according to claim 10, wherein the at least one pathogen is a bacterial species, a fungal species or an arthropod species.

16. The coating composition according to claim 10, wherein the biological agent is at least one biological antagonist that is present in the form of bacterial spores and/or fungal spores located on the surface of the said particles.

17. A seed product of a monocotyledonous plant comprising: (1) a seed of a monocotyledonous plant; and (2) the coating composition as defined in claim 10 in dry powder form on the surface of the seed.

18. The coating composition according to claim 10, wherein the particles of the at least one organic carrier material are electrostatically bound to said one or more biological agents.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Spore loadings of Trichoderma on wheat

EXAMPLES SECTION

(2) Control of Alternaria sp. of wheat (Triticum aestivum) [available from the United Kingdom National Culture Collection (UKNCC)] by means of seed treatments using examples of the antagonists Trichoderma harzianum, Pseudomonas fluorescens and Bacillus subtilis [available from United Kingdom National Culture Collection (UKNCC)]

(3) Alternaria Leaf Blight

(4) Symptoms

(5) Lower most leaves are always the first to show the sign of infection, which gradually spreads to the upper leaves. The disease first makes it appearance as small, oval, discoloured lesions, irregularly scattered on the leaves. The spots became irregular in shape as these enlarge and take up dark brown to grey colour. As the disease progresses, several spots come closer and cover large leaf areas, eventually resulting In death of the entire leaf. A bright yellow marginal zone is sometimes seen around the spots. In case of severe attack, leaf sheaths, awns and glumes are also infected.

(6) Black powdery spores of the fungus cover the lesions at this stage under moist conditions. These spores are disseminated by wind and cause disease on healthy leaves and plants. The disease spreads very fast under warm and humid conditions. Heavily infected fields present a burnt appearance.

(7) Disadvantages of Conventional Seed Treatment

(8) i) Limited dose capacity—The amount of pesticide that can be applied is limited by how much will actually stick to the seed. ii) Limited duration of protection—The duration is often short due to the relatively small amount of biological agent (e.g. chemical) applied to the seed, dilution of the biological agent as the plant grows, and breakdown of the biological agent. iii) Limited shelf life of treated seed—Producing excess treated seed is undesirable because the shelf life of treated seed may be limited. Surplus treated seed cannot be sold for grain.

(9) All three of these limitations may be overcome or significantly reduced through the inclusion of carnauba wax particles as a carrier for a biological agent, in this case dormant microorganisms that are applied to seeds. Under favourable conditions, the microorganisms grow and colonize the exterior of the developing seed or seedling. Biological agents may help in reducing seed decay, seedling diseases, or root rot.

(10) The following tests are performed to examine the potential effect of the inclusion of carnauba wax particles.

(11) Phase One—Isolate Cultures

(12) 1. Culture Maintenance

(13) Records are kept with each isolate sub-culture being assigned an accession number. All plates and slides relating to that sub-culture are labelled with an accession number.

(14) In addition, permanent lactophenol (LP) mount slide are made from each of the original cultures and file for reference purposes

(15) No more than three generations of sub-culture occur before passaging through a living host and re-isolating in order to maintain the fitness of the organism.

(16) Sub-cultures are stored for future use on Potato Dextrose Agar (PDA) at 4° c.

(17) Each isolate is assigned an accession number and sub-cultures are labelled with that number.

(18) DNA is extracted for identity verifications and stored at −20° C. A reference sample of the pure culture is stored on glycerol at −20° C. Upon completion of the experiment DNA identification of the culture is repeated to confirm that the organism has not mutated during the course of the work.

(19) 2. Culturing of the Causal Agent

(20) Isolation of a pathogenic fungus from diseased tissue into pure culture is one of the standard techniques in identifying and describing a disease. It is an essential step in proving the pathogenicity of previously un-encountered organisms.

(21) Techniques Commonly Involve:

(22) a. Surface-sterilisation treatment b. Plating (possibly on selective medium) of samples of diseased tissue, with appropriate precautions. c. Sub-culturing to get pure cultures.
3. Purification of Cultures

(23) Small disinfected root pieces of an artificially inoculated plant are cultured on water agar. The fungal colonies that appear most frequently are likely the target pathogen. Several saprophytes may also be present in infected plant tissues and they may grow into the medium with the principal pathogen. Routine surface-sterilisation consists of wiping the tissue with (or immersing in) 0.1% solution of sodium hypochlorite (NaOCl or sometimes referred to as “NaClO”) followed by rinsing with sterile distilled water. To obtain a pure culture of the pathogen, a small sample is taken from the growing edge of a colony with a flamed loop or scalpel and streaked over the surface of a pre-poured plate of PDA. The inclusion of chloramphenicol (a bacteriostatic anti-microbial) at 30 mg/l reduces the risk of bacterial contamination. As the streak progresses over the agar, fungal spores are separated until single spores are obtained from which separate colonies will grow.

(24) Repeat this procedure until pure cultures are obtained.

(25) 4. Single Spore Isolation

(26) Single spore isolations are important to investigate pathogenic variability. An inoculum of spores is placed in a tube containing 10 ml of sterile water. This spore suspension is streaked along a marked line on the surface of a thin tap water agar medium, and incubated at 22° C. After 24 hr incubation, select germinated spores using a stereoscopic microscope and transferred one spore at a time to another agar plate.

(27) 5. Slide Preparation for Microscopic Examination and Reference

(28) Identification of the pathogen: the tissue may be sectioned or surface scraped and then mounted in water/lactophenol. Fungal structures seen macroscopically may be separated from the host tissue to be examined and identified. Identification depends on spore formation and therefore infected material will be incubated in a moist environment overnight prior to examination in order to encourage sporulation. Cotton blue stain will be added to the lactophenol in order to highlight fungal structure. The specimen will be placed in a drop of satin on a glass slide and gently warmed by passing through a low flame for a few seconds before mounting in lactophenol.

(29) Whole mount sections can be cleared and stained for ease of identification using the following method:

(30) Leaf disks are made clear by heating them in tubes in lactophenol until clear (up to 20 minutes), without boiling. Stain by heating in 0.5% cotton blue in lactophenol on a slide for 5-10 minutes. Rinse thoroughly in lactophenol and mount in the same.

(31) 6. Growth and Media

(32) Sub-cultures are assessed for growth and germination at a range of temperatures, 13.5° C., 18° C. and 22.5° C. A range of media is examined for suitability. Whilst PDA is generally suitable for most fungal species it has been found that use of a low nutrient agar, such as tap-water agar, reduce prolific growth and can encourage sporulation. Therefore PDA, tap-water agar, and a selective media from literature, Czapek's Dox agar (Dawson (1962) Saboutaudia 1. 214-219), are included within the assessment trials.

(33) A 5 mm diameter disk is cut from the margin of an actively growing culture using a flamed cork borer. This is placed upside down in the centre of the pre-poured media plates. Five replicates are made for each media type and temperature (45 plates in total). Complete randomisation will be applied to plates in each incubator. Plates are observed until one culture succeeds in completely covering the plate in any one media. At this point the following measurements are taken: fungus colony diameter, colour and margin. In addition, the level of sporulation is recorded.

(34) Five 5 mm disks are cut from each plate using a flamed cork borer and suspended in 20 ml of distilled water (+0.05% Tween 20®). The sample is then sonicated for 2 minutes to release the spores and then vortexed to aid the formation of a uniform spore suspension. Samples are assessed for spore concentration using an Improved Neubauer haemocytometer using standard counting methodology.

(35) The mean for each media type is calculated and ANOVA is applied to examine the results for significant differences.

(36) Phase Two—In Vitro Studies:

(37) 1. Screen Microorganisms and Carnauba Wax to Determine Interactions

(38) In order to explain effects observed the microorganisms, pathogens and antagonists, are screened against carnauba wax to identify any carrier only effect. This will enable the determination of treatment effect as well as any synergy occurring as a result of the use of using an antagonist with carnauba wax particles. a. Plates of appropriate media are used based on the findings of the experiment above. Air-milled carnauba wax is sterilised using the autoclave and then ground using a twin blade mill, producing particles with an approximate VMD of The sterilised media is then cooled to 50° C. (molten stage). The carnauba wax is then incorporated into the media. Two concentrations of carnauba wax are tested; 1 g/l and 10 g/l. A 5 mm diameter disk is cut from the margin of an actively growing culture using a flamed cork borer. This is placed upside down in the centre of the pre-poured media/carnauba wax plates. Five replicates will be made for each concentration and incubated at the optimum temperature for growth/sporulation (as determined in previous experiment). Growth rates and characteristics are compared to the controls using data from the Growth and Media experiment above.
Differences Will be Analysed Using ANOVA. b. Disks of the pathogen and antagonists are dusted with different carnauba wax treatments and put on appropriate media. The carnauba wax particles need to be free of microorganisms to be able to carry out this experiment. Growth of treated and untreated organisms are compared.
2. Investigate Antagonist Action Against Pathogens
i. Effect of Antagonists on Viability of Alternaria sp. Mycelium (In Vitro Assay I)

(39) All antagonistic isolates are tested in a dual culture assay against pathogenic fungi on PDA or alternative pre-defined media. Agar plugs of Alternaria sp. and the antagonist isolate to be tested are arranged 7 cm apart on 9 cm agar plates. Inhibition zones and zones of overlapping are assessed after 7 days incubation at 13.5° C., 18° C. and 22.58° C. Where an antagonist overgrows the mycelium of Alternaria sp., the zone of hyphal interaction between both is investigated microscopically (100×). Fungal strains without a microscopically visible effect on mycelium of Alternaria sp. are excluded from further experiments. Furthermore, the viability of Alternaria sp. in the region of interaction is tested by transfer of mycelial discs onto water agar plates 5 days after first contact. The Alternaria sp. mycelium is assessed as viable when the growth of typical hyphae is observed microscopically (100×). Each experiment is repeated three times with three samples per replicate.

(40) ii. Effect of Antagonists on Germination of Alternaria sp. Sclerotia Produced In Vitro (in vitro assay II)

(41) Sclerotia of Alternaria sp. of uniform size are placed on a 6 day old culture (PDA, 20° C.) of the fungal antagonist. After incubation for 14, 28 and 35 days at 20° C., eight sclerotia per replicate (three replicates per antagonist) are transferred from the agar plate onto water agar. Mycelial growth from these sclerotia is assessed under a light microscope (100×).

(42) 3. Confirmation of Pathogenicity

(43) Steps to perform Koch's postulates (Koch 1890, criteria designed to establish a causal relationship between a causative microbe and a disease) a) Describe the symptoms expressed by the diseased crop plants. b) Isolate the suspected pathogen—the same cultures should be isolated from plants with similar symptoms c) Obtain a pure culture and use it to inoculate healthy plant material. d) Observe the symptoms expressed by the inoculated plants—symptoms should be the same as those observed originally in the crop plants. e) Re-isolate the pathogen from the newly diseased material. The culture should be the same as the original purified culture.
i. Indirect Application—Plant Using healthy plants—soil can be inoculated directly using a spore suspension made from a pure agar culture or from a culture grown in flasks. A fungal spore or bacterial suspension can be added post-emergence so that the root system is drenched by the suspension. Plants are then observed over 7 days and symptoms recorded. Koch's Postulates are applied in order to confirm that the symptoms relate to the inoculated pathogen.
ii. Direct Application—Seed Inoculum for preparing spore suspensions is grown on water agar containing sterile seeds. Fungal spores and hyphae or bacterial spore and vegetative growth are scraped from the colony and transfer to sterile water. This spore suspension is then applied to seeds and mixed to ensure a uniform distribution. Seeds are then: Placed on moist filter paper and incubated at optimum growth temperature for 5 days. sown in heat sterilised potting compost and incubated in a propagator at optimum growth temperature for 7 days Symptom expression and germination is recorded for both sets of experiments and Koch's postulates applied
4. Carnauba Wax/Antagonist Co-Location Analysis

(44) A dry powder formulation of spores is produced using a spore separator. Moisture content of the formulation is reduced to below 5% using a dehumidifier and silica beads. Spore concentration is determined using a Neubauer haemocytometer and standardised counting methodology.

(45) Steps in Air Milling in Boyes Micronisation Process (for carnauba wax particles with a VMD of approx. 25 μm and 75 μm)

(46) 1. 2 kg carnauba wax blocks are first kibbled into approximately 4 to 6 mm pieces in a KT Handling Ltd Model 04 kibbler (serial no. 729/C) following the manufacturer's instructions.

(47) 2. The kibbled pieces are then passed through an Apex Construction Ltd Model 314.2 Comminuting Mill (serial no. A21306) and reduced further in size to a range of 250 to 300 um.

(48) 3. The comminuted particles are then passed through a Hosokawa Micron Ltd Alpine 100AFG jet mill (serial no. 168092) following the manufacturer's instructions, setting the mill at a suitable speed (a speed of 8000 rpm for particles having a VMD of 15 μm or at a speed of 2500 rpm for particles having a VMD of 75 μm), with a positive system pressure of 0.03 bar.
4. The grinding air is to be kept to 6 bar, the system rinsing air flow and Classifying Wheel gap rinsing air are both to be set at a minimum of 0.5 bar and no more than 0.75 bar, the cleaning air filter is to register a delta of no more than 5 bar to achieve a final particle size with a VMD of 15 um or 75 μm as required.

(49) Entostat was combined with wheat seed at three loadings (see below).

(50) Two sizes of carnauba wax particle having VMDs of 15 μm and 75 μm, respectively, are examined in combination with the spore formulation at two different ratios (1:3, 2:2). Samples of the carnauba wax/spore mixture are analysed using electron photomicroscopy to determine the co-location effect. Any variation observed is recorded.

(51) In addition, both sizes of carnauba wax referred to, are mixed with a homogenised sample of mycelium and examined as described above.

(52) 5. Carnauba Wax Particle Loading

(53) Carnauba wax particle adhesion to seeds is approximated through the use of photomicroscopy (qualitative) and fluorometric analysis (quantitative). Two sizes of carnauba wax particles (with 1% glo-brite) are used having a VMD of 15 μm and 75 μm, respectively. Four combinations: Two ratios of carnauba wax/spore formulation, together with one mycelial and a vehicle control (carnauba wax only), makes a total of eight treatments. Treatments are applied to 10 g of seed and replicated three times. Three subsamples are taken from each replicate and the mean used in analysis.

(54) For fluorometric analysis three 1 g samples are each added to 5 ml of ethanol and sonicated to aid the release of the carnauba wax particles from the seeds. Samples are analysed using a Perkin Elmer L55 Fluorometer (Perkin Elmer, Ma, USA). Statistical analysis of variation between treatments is performed using ANOVA.

(55) Seed size and architecture varies greatly between crop species and this influences application rates and method. A homogeneous mix is attained through tumbling seed and carnauba wax formulation in a cylinder, adapted to produce lateral mixing/tumbling through the inclusion of angled interior vanes, placed on a Wheaton roller for 5 minutes.

(56) Phase Three—In vivo:

(57) Alternaria sp., together with the most successful antagonist model are used in a series of in vivo experiments. The basic design is a split-plot experiment with temperature being the main plot factor (13.5° C., 18° C. and 22.5° C.) and carnauba wax/antagonist ratio (3 treatments:2× spore, 1× mycelial) being the sub-plot. Four homogeneous mixes of each treatment are prepared using the method described above and these represent the replicates.

(58) Treatments:

(59) 1) Application rate 1—7.5×10.sup.6 conidia kg.sup.−1 2) Application rate 2—7.5×10.sup.8 conidia kg.sup.−1 3) Application 3—Mycelia 4) Control 1—Vehicle control (Carnauba wax only) 5) Control 2—no treatment
Mixes (true replicates): A, B, C, D
Subsamples of each mix: α, β, γ
Mixes and treatments are arranged according to a Randomised Block design.
Pot Studies

(60) Each temperature (growth chamber) contains 60 plant pots.

(61) Treated seed is sown in accordance with supplier's recommendation. Soil/compost (1:1 John Innes No. 2 and peat compost) is heat sterilised prior to inoculation with 10 ml of Alternaria sp. spore suspension and thoroughly mixed before sowing.

(62) Plants are placed in the growth chambers for a period of 21 days with observations of symptom expression made every 48 hours post emergence. Water is applied through capillary matting twice daily.

(63) After 21 days plants are removed from their pots and the following assessment measurements taken:

(64) % germination % pre-emergence damping off % post-emergence damping off Root weight Shoot weight

(65) In addition, symptom expression is assessed based on a damage scale.

(66) Means of the measurements taken from the subsamples α, β, γ are compared for each treatment using ANOVA.

(67) Samples are taken from 5 plants exhibiting symptoms and Koch's Postulates applied to confirm the causal organism (by comparison to the reference slide of the master culture). The experiment is repeated.

Second Example

(68) Control of Pythium graminicola [United Kingdom National Culture Collection (UKNCC)] on wheat (Triticum aestivum) by means of seed treatments using fludioxonil.

(69) Experimental Design—as per the Pot Study in Example 1, above

(70) Carnauba wax is melted using copper pans. During cooling fludinoxonil is added at 1% of the mass of the carnauba. This mixture is allowed to solidify before chipping and processing through an air mill as described above, with the exception that the speed is set at 6000 rpm to produce particles with a VMD of 25 μm.

(71) Treatments for the Pot Study

(72) Control 1—Vehicle control (Carnauba wax only)

(73) Control 2—no treatment

(74) Treatment 1—1% fludinoxonil carnauba wax at 10 g per kg of seed

(75) Treatment 2—1% fludinoxonil carnauba wax at 3.2 g per kg of seed

(76) Assessment and analysis as with previous Pot Study

Third Example

(77) Relating to:

(78) Control of Agriotes Mancus spp. (Coleoptera: Elateridae), or Wheat Wireworm, (the larval form of the click beetle) that preys on wheat (Triticum aestivum), by means of seed treatments using thiamethoxam.

(79) Early-season wireworm damage consists of hollowed-out seeds where larvae have entered during germination. Seedling plants also can be injured or killed by larvae tunneling into the plant below the soil line. Occasionally, wireworms bore into the stalks of larger plants and tunnel in a few inches, but the damage is not significant.

(80) Experimental Design—as Pot Study Above

(81) Carnauba wax is melted using copper pans. During cooling thiamethoxam is added at 1% of the mass of the carnauba. This mixture is allowed to solidify before chipping and processing through a mill as described above (speed set at 6000 rpm) to produce particles with a VMD of 25 μm.

(82) Treatments for the Pot Study

(83) Control 1—Vehicle control (Carnauba wax only)

(84) Control 2—no treatment

(85) Treatment 1—1% thiamethoxam carnauba wax at 4.2 g per kg of seed

(86) Treatment 2—1% thiamethoxam carnauba wax at 1.3 g per kg of seed

(87) Empty pots are lined with a nylon mesh screening material before filling with potting soil. A wire frame is constructed and the nylon meshed tied off over the frame to provide a caged experimental arena designed so that the insect cannot escape the treated area.

(88) Seeds are allowed to germinate for three days before adding five 3.sup.rd instar larvae to the soil surface of each pot before resealing the mesh cage.

(89) Observations are made over 21 days.

(90) Plants are assessed for:

(91) % germination Damage Root weight Shoot weight

(92) The procedures detailed within Example One are followed to examine the antagonistic effect of Trichoderma harzianum [United Kingdom National Culture Collection (UKNCC)], Pseudomonas fluorescens [UKNCC] and Bacillus subtilis [UKNCC] on Fusarium sp., a fungal pathogen of Rice (Oryza sativa).

(93) The procedures detailed within Example One are followed to examine the antagonistic effect of Trichoderma harzianum [United Kingdom National Culture Collection (UKNCC)], Pseudomonas fluorescens [UKNCC] and Bacillus subtilis [UKNCC] on Colletotrichum graminicola, a fungal pathogen of Sorghum (Sorghum bicolor).

(94) The procedures detailed within Example Two are followed to examine the effect of metalaxyl on Pythium sp., a fungal pathogen of Rice (Oryza sativa).

(95) The procedures detailed within Example Two are followed to examine the effect of prochloraz on Rhizoctonia sp., a fungal pathogen of sorghum (Sorghum bicolor).

(96) The procedures detailed within Example Three are followed to examine the effect of thiamethoxam on the White Grub (Phyllophaga crinite), an insect pest of Sorghum (Sorghum bicolor).

(97) The procedures detailed within Example Three are followed to examine the effect of imidacloprid/beta-cyfluthrin on Rice Seed Midges (Cricotopus sylvestris), an insect pest of Rice (Oryza sativa).

(98) Suppression of Causal Agents of Fungal Disease in Wheat (Triticum aestivum) Using a Seed Coating Comprised of Trichoderma sp. and Carnauba Wax Particles

(99) The potential for Trichoderma sp. (Ascomycota) as a biocontrol agent in the defence against plant pathogens is known.

(100) Trichoderma hyphae are capable of penetrating the hyphae of other fungi and extracting nutrients from within, resulting in the suppression and eventual death of the host. Trichoderma exhibits rapid mycelial growth and is capable of out-competing other fungi for nutrients.

(101) There are several commercially available formulations of Trichoderma marketed as crop protection products. These are commonly supplied as a wettable powder formulation and applied to the area of cultivation as a drench. The disadvantage of this form of application is that it is necessary to treat the entire cultivation area, whereas it is the region immediately surrounding the seed or plant that requires the treatment. The larger the number of conidia delivered to this area the greater the level of control they are able to impart. Therefore a targeted application system able to deliver sufficient conidia to the required area offers a distinct advantage in the use of Trichoderma over conventional applications.

(102) Experimental Aim: To Assess the Potential Use of Entostat as a Seed-Coating Technology for the Delivery of Beneficial Microbes

(103) Methods

(104) Steps in Air Milling in Boyes Micronisation Process (for carnauba wax particles with a VMD of approx. 10 μm)

(105) 1. 2 kg carnauba wax blocks are first kibbled into approximately 4 to 6 mm pieces in a KT Handling Ltd Model 04 kibbler (serial no. 729/C) following the manufacturer's instructions.

(106) 2. The kibbled pieces are then passed through an Apex Construction Ltd Model 314.2 Comminuting Mill (serial no. A21306) and reduced further in size to a range of 250 to 300 um.

(107) 3. The comminuted particles are then passed through a Hosokawa Micron Ltd Alpine 100AFG jet mill (serial no. 168092) following the manufacturer's instructions, setting the mill at a speed of 12500 rpm, with a positive system pressure of 0.03 bar.

(108) 4. The grinding air is to be kept to 6 bar, the system rinsing air flow and Classifying Wheel gap rinsing air are both to be set at a minimum of 0.5 bar and no more than 0.75 bar, the cleaning air filter is to register a delta of no more than 5 bar to achieve a final particle size with a VMD of 9.7 μm.

(109) Entostat was combined with wheat seed at three loadings (see below). 1. Baseline data: seed coating techniques 1.1. Seed Coating. Trichoderma harzianum (containing 7.75×10.sup.9 colony forming units g.sup.−1 Sylvan Bio, Loches, France) with a germination percentage of 95% was applied to wheat (var. Hereward, Herbiseeds, Twyford, UK) using carnauba wax particles with a VMD of 9.7 μm. A target loading was set at 10.sup.5 conidia per seed based on information obtained from literature. Carnauba particles were mixed with the dry conidia powder at different ratios and applied 0.01 g (0.2% by mass) directly to dry seed, 5 g of seeds per concentration. For each concentration, four batches of 10 seeds were used for evaluation of conidia loading. Conidia to carnauba ratios used were: 100% Conidia, 50% Conidia, 25% Conidia and 9% Conidia with the remainder in each case being made up of carnauba wax particles. 1.2. Enumeration. Direct enumeration to determine conidia loading of seeds was done through the use of a haemocytometer (Improved Neubauer, Hawksley, Lancing, UK). Inoculum: Preparation of suspension. Propagules are usually formulated in a water carrier, although those with hydrophobic cell walls (such as Trichoderma) are not readily suspended in water. To uniformly suspend hydrophobic propagules in water it is necessary to sonicate and/or use mechanical suspension methods. Mechanical suspension of propagules using micropestles provides good suspension of conidia in water without causing damage to cells. A surfactant may also facilitate suspension of propagules (Tween20 at 0.05%). To suspend hydrophobic conidia, harvested conidia are placed in a 1.5 ml microcentrifuge tube, ≈0.5 ml of sterile water is added to the tube, the micropestle is inserted into the tube, and the conidial mass is gently agitated with the micropestle by hand (prevents liberation of conidia into air). The micropestle is the attached to the motor (e.g. Kontes, Argos pellet pestle motor) and the suspension is vigorously agitated while moving the pestle in and up and down, and side to side motion, circa. 30 seconds. Since the haemocytometer method does not distinguish between viable and non-viable propagules, it is necessary to determine spore viability so that doses can be prepared on the basis of viable propagules. Seed washes and enumeration of Trichoderma loadings were done on 4 batches of seeds per treatment. Inoculum was washed from seeds by placing into 1 ml sterile 0.05% Tween.sup.20 (or substitute—similar non-ionic surfactant/dispersal agent) in a Eppendorf tube and vortexing for 30 seconds to remove conidia from the seed surface. Samples were then sonicated for two minutes to break up any conidial clumping. Counts obtained were used to calculate the mean conidia loading of seed coated with the various treatments. Results obtained using 100% conidia powder were used as a benchmark and the conidia/carnauba combination powders compared against it as a determination of efficiency of loading. Confirmation of conidial viability was achieved by dilution plating on Trichoderma Specific Media (TSM) (see below). A dilution series was set up and duplicate plates inoculated from the series. Colony Forming Units (CFU) counts were made after 7 days, allowing inoculum levels on seeds to be quantified. In addition, fresh, unused conidia were plated to provide a comparison of before and after seed application. Germination percentage was also measured. A satisfactory density of conidia was obtained by spreading approximately 10.sup.6 conidia in 100 μl on the media in a 9 cm petri dish. Conidia were incubated in the dark at 25° C. for five days, and the area to be observed was then fixed using lactophenol. Phase contrast microscopy using an inverted compound microscope enabled sufficient examination of the conidia. Conidia were considered viable if germtube lengths were two times the diameter of the propagule in question. Numbers of germinated and non-germinated conidia in arbitrarily-selected fields of view or in parallel transects, defined with an ocular micrometer, were counted. A minimum of 300 conidia were counted to provide an accurate estimate. It is desirable to determine the viability of propagules on replicate cultures and at various positions on the same plate. This allowed calibration of the seed-coating techniques to obtain similar levels of Trichoderma loadings on the seeds for each coating method. 1.3. Seed Germination. One batch (5 seeds) of seeds from each treatment was placed on seed test paper (Whatman 181) in a 9 cm Petri dish. Dishes were sealed with Parafilm and held at 20° C. for 7-10 days and germination rate determined. This was repeated with untreated seed.
Trichoderma Selective Media (adapted from Williams, Clarkson et al 2003) was prepared as follows:
For 1000 ml
Basal Medium Ingredients:

(110) TABLE-US-00001 0.2 g MgSO.sub.4 3.0 g glucose 0.9 g K.sub.2HPO.sub.4 0.15 g rose bengal 0.15 g KCl 20 g agar 1.0 g NH.sub.4NO.sub.3 950 ml distilled water
Basal Medium Process

(111) Mix liquid ingredients with all solid ingredients, except the agar in a 1 L Erlenmeyer flask. Add the 20 g agar and stir or shake. Plug with cotton wool and cover with foil. Autoclave.

(112) TABLE-US-00002 Biocidal Medium (per liter) 0.25 g crystallized chloramphenicol 0.2 g quintozene 0.2 g captan 1.2 ml propamocarb (Previcur) 50 ml sterile distilled water
Seed Weight

(113) Used as a measure of the homogeneity of the seed batch. Eight replicates of 25 seeds are weighed and the coefficient of variation (Cv) recorded. This coefficient should not exceed a value of 5. If it does then the procedure is repeated and the mean of all 16 samples used to calculate the number of seeds per gram.

(114) TABLE-US-00003 Crop Mean Weight (g) SD Cv TGW (g) Wheat 1.258 0.059 4.678 50.305
Results
Direct Enumeration Counts Using Haemocytometer

(115) Initial Spore Density of Trichoderma harzianum dry spore preparation (at 5% moisture content), determined using haemocytometer, was 7.75×10.sup.9 spores g.sup.−1 (n=4,±2.6×10.sup.7 95% CL).

(116) Spore Counting of Seed Wash

(117) TABLE-US-00004 Variable Spore % N Mean SE Mean SporeCount 9 4 300750 11499 25 4 757750 21453 50 4 1062500 18875 100 4 2145000 109278 *10.sup.5 target spores per seed

(118) There was a clear and statistically significant difference between the mean spore counts per seed achieved by the different treatments as determined by one-way ANOVA (F(3,12)=190.83, p=<0.001). All treatments exceeded the target of 10.sup.5 spores seed.sup.−1.

(119) TABLE-US-00005 Mean Spore *Expected As a % **As a % % Count Spore of 100% of t p Spores Seed.sup.−1 Count Treatment Expected value value 100%  905250 n/a n/a n/a n/a n/a 50%  1062500 1072500 50%  99% −0.53 0.633 25%* 757750 536250 35% 141% 10.32 0.002 9% 300750 193050 14% 156% 9.37 0.003 *Expected Spore Count is calculated from the mean spore count achieved by the 100% Treatment, assuming a perfect distribution. Therefore the 50% Treatment would be expected to result in half the spores of the 100% Treatment, and so on. **Essentially a measure of improvement in spore adhesion efficiency.

(120) The addition of Entostat above 50% appears to improve the efficiency of spore adhesion to seed as the actual mean counts significantly exceed the expected results based on the 100% spore treatment (t-test).

(121) Germination Determination

(122) Mean conidia germination (from a sample of 300)

(123) Fresh conidia—276.50±10.85, n=4

(124) Seed wash conidia—275.25±6.02, n=4

(125) There was no statistically significant difference between the viability of fresh conidia and those washed from seeds as determined by one-way ANOVA (F(1,6)=0.04, p=0.847).

(126) Enumeration Estimate from CFU Counts

(127) Comparison of Haemocytometer and CFU (corrected for dilution) counts

(128) TABLE-US-00006 Grouping Treatment N Mean SE Mean (using Tukey method) 100CFU 4 2150000 83964 A 100Haemo 4 2145000 109278 A 50CFU 4 1060000 12910 B 50Haemo 4 1062500 18875 B 25CFU 4 787500 15058 C 25Haemo 4 757750 21453 C 9CFU 4 303250 9059 D 9Haemo 4 300750 11499 D Mean that do not share a letter are significantly different.

(129) There was a statistically significant difference between groups as determined by one-way ANOVA (F(7,24)=205.95, p=<0.001). A Tukey post-hoc test revealed significance was as a result of differences in the spore % rather than the counting method applied.

SUMMARY

(130) Wheat seed can be coated with Trichoderma spores in excess the target 10.sup.5 spores seed.sup.−1 for all treatments.

(131) Use of Entostat at a ratio greater than 1:1 increases the efficiency of spore delivery as a result of a reduction in wasted or lost spores.

(132) The germination viability of the spores was unaffected by their use as a seed coating.

(133) Enumeration through direct counting of spores using a haemocytometer or through the use of CFU counting gives statistically similar results and therefore either method may be used once germination viability has been proved unaffected by the treatment.

(134) The described method for wheat as provided above is used to assess the delivery efficiency of spores by Entostat to seeds of barley, rye, oats, maize and rye grass. Similar or better results are obtained, depending on loading and seed size.

(135) Effects of Seed Coating on Disease Suppression

(136) Seeds are coated with Trichoderma using water or Entostat to achieve loadings of ca. 10.sup.5 and 10.sup.6 CFUs seed.sup.−1. Water treatments are suspensions of spores in sterile water in which the seed samples are soaked for one hour. Seeds are then dried back, a likely commercial scenario, or sown wet coated. Entostat is applied at ratios of 3:1, and 9:1, Entostat to spores respectively. Seed treatment methods are then compared for their ability to protect germinating wheat seedlings from Gaeumannomyces graminis, the causal agent of take-all disease in wheat.

(137) Inoculation of seeds with Trichoderma. Wheat seed cv. Hereward is inoculated as follows (target concentration per seed):

(138) 1) Trichoderma at 10.sup.5/seed using a water suspension (wet coating) 2) Trichoderma at 10.sup.6/seed using a water suspension (wet coating) 3) Trichoderma at 10.sup.5/seed using a water suspension (dry coating) 4) Trichoderma at 10.sup.6/seed using a water suspension (dry coating) 5) Trichoderma at 10.sup.5/seed using Entostat at 3:1 6) Trichoderma at 10.sup.6/seed using Entostat at 3:1 7) Trichoderma at 10.sup.5/seed using Entostat at 9:1 8) Trichoderma at 10.sup.6/seed using Entostat at 9:1 9) No Trichoderma, water only 10) No Trichoderma, Entostat only 11) Seed only

(139) Enumeration. Trichoderma is quantified using standard dilution plating methods on Trichoderma specific media. This confirms CFU loadings per seed for treatments 1-8. Dilution platings are carried out in duplicate.

(140) Gaeumannomyces Bioassay

(141) Inoculum preparation—Gaeumannomyces sp., known to be pathogenic on wheat, barley, rye, oats and turf grass, is grown on PDA plates from stock cultures, and incubated at 20° C. to produce actively growing colonies. Agar plugs are removed from the plates and used to inoculate sterilised (autoclaved at 121° C. for 20 mins) John Innes No. 2 potting mix (80% moisture content; 60 g) mixed with potato cubes (2 mm.sup.2, 25 g) in 500 ml Erlenmeyer flasks. Flasks are incubated at 20° C. for 14 days. Inoculum levels in the medium are quantified using a dilution plating method.

(142) Effectiveness of seed treatment on Gaeumannomyces. Seeds are sown into individual cells of seed trays containing Gaeumannomyces-inoculated medium (approx. 15 ml/cell). Four replicate batches of ten seeds per treatment are planted into the cells. Once sown, the trays are placed in a plant growth chamber (Weiss Gallenkamp Fitotron SG120) at 20° C. with ca. 16 h lighting. Cells are bottom watered. The number of seedlings surviving are recorded every 3 days for 21 days.

(143) Time to emergence, percentage successful emergence and percentage plants expressing symptoms are recorded and the results analysed. Differences in Entostat treated seed and untreated seed are observed.