AU593842B2 - Method of protecting useful plants from diseases caused by soil-borne and seed-borne pathogens by treating seeds with cultures of microorganisms - Google Patents

Description

i I i i I 7 FORM 10 SPRUSON FERGUSON COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFI5T9 3 8 4 2

(ORIGINAL)

FOR OFFICE USE: Int. Class (Ct, (C I r.

1 ft 'tt I CC tr C (f r Class Complete Specification Lodged: Accepted: Published: his document contains the amiendrnents made under Section 49 and is correct for printing.

Priority: Related Art:

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Name of Applicant: Address of Applicant: Actual Inventor(s): Address for Service: CIBA-GEIGY AG Klybeckstrasse 141, 4002 Basle, Switzerland DANIEL GINDRAT and DANIEL WALTHER Spruson Ferguson, Patent Attorneys, Level 33 St Martins Tower, 31 Market Street, Sydney, New South Wales, 2000, Australia i' Complete Specification for the invention entitled: "METHOD OF PROTECTING USEFUL PLANTS FROM DISEASES CAUSED BY SOIL-BORNE AND SEED-BORNE PATHOGENS BY TREATING SEEDS WITH CULTURES OF MICROORGANISMS" The following statement is a full description of this invention, including the best method of performing it known to us SBR:ALB:195U

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Case 5-15520/1+2/= Method of protecting useful plants from diseases caused by soilborne and seed-borne pathogens by treating seeds with cultures of microorganisms it i The present invention relates to a method of protecting useful St, plants, especially sugar beet and cotton plants, from diseases caused by soil-borne ae -al n bed-barno\microorganisms. The method comprises providing the seeds with a protective coating which S, contains an effective amount of fungus spores, in particular ttt ascospores of Chaetomium globosum, mycelium, bacteria or culture extracts thereof. The invention also relates to the seeds coated with Chaetomium globosum ascopores, and to a process for the preparation thereof.

9EIt Seeds shall be understood in the context of this invention as meaning in particular the parts of plants necessary for propagation, for example grains, seeds, tubers, cuttings and shoots.

The germinating plantlet is often attacked by soil-borne pathogenic microorganisms that cause the plants to rot and die, whereby severe damage is caused to agriculture. Such pathogens are normally controlled with chemical fungicides.

AD Attempts have also been made to use microorganisms themselves to protect plants against attack by pathogens. Interesting as such attempts have been, suitable methods have been unable to gain acceptance in actual practice because industrially produced fungicides have proved more effective and easier to handle.

C i 4J 2 The present invention is based on the surprising observation that it has been possible in in vitro and in field tests to match and even surpass the protective action of seeds of useful plants, especially of sugar beet and cotton, coated with commercially available fungicides by dressing seeds with a coating that contains ascospores of Chaetomium globosum. The spores used are simple to culture and are not more susceptible to drying out than the seeds coated therewith. The spores can be mixed in simple manner with a seed dressing. They are also no more difficult to handle than a chemical IC) fungicide. The spores can be cultured and harvested using simple rt equipment and with little effort.

t Sugar beet plants (Beta vulgaris) are attacked in the soil by a variety of microorganisms, among which the most important are pathogenic fungi such as Pythium ultimum, Rhizoctonia solani, SAphanomyces cochlioides, Phoma betae, Fusarium sp., and Botrytis sp.. These pathogens infect the tissues of the roots and stalk, resulting in breakage and, ultimately, in rot of the plantlet.

The sugar beet seeds are flat, pentagonal stars which tend to become entangled with one another. For trouble-free mechanical sowing, they O0 are coated and pelleted by the seed dealer. The seed coating routinely contains a chemical fungicide or mixture of fungicides.

Cotton plants (Gossypium hirsutum) are likewise infected by the fungi Pythium ultimum and Rhizoctonia solani, along with Thielaviopsis basicola and Fusarium-spp. These are usually large-scale crops which are intensively treated with agrochemicals, as diseases and crop losses result in financial loss that cannot be compensated for by yields of other crops. The treatment of the crop and/or of the seeds with fungicides is a routine matter in cotton growing.

Chaetomium globosum belongs to the fungi of the class Ascomycetes.

3O It is the most widespread species of Chaetomium. The fungus is found in the soil and, in particular, in the root zone of different 1 T: 3plants. It is a cellulose saprophyte which is found on plant residues. The ability to degrade cellulose and other polysaccharides has been studied but not utilised technically.

The use of Chaetomium globosum for controlling plant pathogens has been described in other contexts. Thus, for example, Tveit and Wood have described the control of Fusarium nivale blight in oats in Ann.

appln. Biol. 43, 538-552 (1955); and the control of corn root infection caused by Fusarium roseum has been described by Chang and Kom.nedahl 4 n Phytopathology 58, 1395-1401 (1968) and by Kommedahl and Mew in Phytopathology 65, 296-300 (1975). F_.ally, Heye and Andrews describe the control of apple scab caused by Venturia inaequalis in Phytopathology 73, 650-654 (1983).

The successful use of Chaetomium globosum for protecting seeds of sugar beet and cotton is novel.

Chaetomium globosum can be readily cultivated even at room temperature in a malt or cellulose-containing culture medium that contains trace elements in a neutral to acid pH range.

The mycelium forms rapidly as a tissue mat that adheres to a solid culture medium or, if it has been cultured on a filter paper placed RO on the culture medium, also adheres to a filter paper culture. The perithecia that contain ascospores then form on the hyphae and impart a black colour to the substrate. A mycelium that carries no perithecia forms in liquid shake cultures. On the filter paper cultures, the perithecia are isolated by scraping them off the filter paper and they are then dried and processed by grinding them to a powder that consists chiefly of free ascospores and mycelium and perithecia fragments. In liquid cultures, the mycelium is isolated by filtering the medium and the isolate is then comminuted in a high-speed rotary propeller mill.

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4 7 I -4 The resultant powder consisting of ascospores and pieces of hyphae is stable and can be stored in the temperature range from 100 to and at a relative humidity from 20 to 60 Even spores that are stored for some considerable time germinate within hours when they come in contact with moist soil and/or a nutrient medium.

The seeds of useful plants are wetted in known manner with n liquid coating material that can be dried. Then, during the drying procedure when they are still tacky, the seeds are contacted with the ascospore powder, e.g. by shaking or rolling the seeds over a layer I0 of powder which has been applied to a surface and finally dried until the coat is completely dry.

Coating compositions for the seed dressing are suitable solutions.

These solutions may contain water or an organic solvent, and the material forming the protective coat may be organic or inorganic.

Typical examples of such solutions are: r Cl V t 't V Vr I V V Il 18 parts of 9 73 1O 50 parts parts parts ;i; parts of 1-2 parts of 200 parts of 1-2 parts of 100 parts of a polyurethane prepolymer containing free isocyanate end groups (No. W. 23 091, Pittsburg Plate Glass Co.) polyketimine (No. W. 23 092, Pittsburg Plate Glass Co.) acetone a high molecular polyvinyl acetate homopolymer (S-6930, H.E. Fuller Co.) water methyl cellulose water xanthane water 15-30 parts of gum arabic 100 parts of water.

it r 5 Coating compositions containing clay particles, alginates and further formulation substances are also employed. Preferable solutions which can be dried contain water, methyl cellulose and gum arabic or contain water and iron phosphate.

The seeds are moistened or sprayed with the solutions and then dried in a stream of air on a sieve or in a fluidised bed drier and dusted while still tacky with the ascospore powder. The number of spores applied to each plant should be 10 to 6 x 10 most preferably 1.5 x 10 5 to 6 x 5 to be fully effective. The spore density is determined by dissolving a number of seeds in water and analysing the suspension with a colorimeter or a hematometer.

#i The seeds coated with the spores can be stored just as well as untreated seeds. During germination they are protected against infections caused by soil-borne pathogens.

15 The culturing of Chaetomium globosum and the harvesting of the ascospores is extremely simple and can be effected without technical aids even on a small scale. This can be of particular interest whenever the use of chemical fungicides is dispensed with for one reason or another.

Chaetomium globosum grows virtually everywhere that cellulose or polysaccharides and moisture are available to it. Culturing was possible in different culture media and in the temperature range from 10 0 -30 0

C.

Examples of liquid and solid culture media are: 2 parts of malt extract (Oxoid a registered trade mark) (adjusted to pH 5.3) 100 parts of water (culturing for 4 days at 24 0 C with slow speed stirrer).

1 part of glucose 0.3 part or yeast extract (Difco (a registered trade Mark)) 2 parts of agar (Sigfried (a registered trade mark)) 100 parts of water

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.L52- 6 1 part of cellulose (Merck, microcrystalline) 2 parts of agar 0.3 part of yeast extract (Difco) eo e-red Tr 100 parts of water 1 g of KHaP0 4 (Czapel-Dox) g of MgS04 g of KC1 2 g of NaNO 3 0.1 g of FeS04 IO 20 g of glucose Swater to make up 1 litre 2 parts of male extract (Oxoid) e TT a 2 parts of agar (Sigfried) (Ren e~--rea r--o Y 100 parts of water 1 g of KH 2 PO4 g of MgS0 4 2 g of KNO 3 2 g of CaC03 g of glucose S> 0.1 g of yeast extract 12 g of malt extract (corn steep liquor) water to make up 1 litre The mycelium which is densely populated with perithecia is decanted or isolated from the culture after 4-6 weeks and dried.

The culture of ascospores is also obtained by soaking filter paper with a suspension containing 10 -10 5 spores per ml in a culture broth and then culturing in the temperature range from 10 0 -25°C in a

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petri dish at relatively high humidity. After about 4 weeks the filter papers are dried and the fungal biomass can be scraped off O with a spatula.

7- The invention is illustrated by the following non-limitative Examples.

Example 1: Culturing ascospores of Chaetomium globosum Petri dishes, to each of which has been added a filter paper as source of cellulose, are half-filled with a nutrient solution consisting of malt (2 agar (2 The nutrient solution is then I inoculated with a few drops of a suspension of soil infected with Chaetomium globosum. The petri dishes are kept in climatic chambers at 22 0 C. A mycelium forms on the culture, followed after about days by the formation of perithecia which are visible as black dots on the mycelium. After 4 weeks, a number of perithecia are removed from the culture and processed to a suspension which Scontains 104-105 ascospores per ml. Filter papers are then soaked with this suspension, placed on malt extract nutrient media in petri dishes and cultured for 4-6 weeks at 22°C in a climatic chamber. The filter papers are overgrown during this time with mycelium and perithecia. The filter papers are dried and the mycelia and spores are scraped off with a spatula and the fungal biomass is ground or AO comminuted in a mixer at 500 rpm, to give a dark powder consisting of spores and fragments of hyphae that is suitable for seed coating.

A round filter paper of 8.7 cm diameter yields on average 120 mg of powder consisting of 108-109 ascospores and fragments of hyphae.

Example 2: Coating sugar beet seeds with a layer of ascospores of Chaetomium globosum Adhesive solutions are prepared consisting of methyl cellulose 0.5 part 1 part gum arabic 15.0 parts 30 parts water to make up 100 parts by volume.

2 i' 8 These solutions are sterilised for 20 minutes in an autoclave at 120°C and then blown through a jet into the air stream of a fluidised bed drier containing sugar beet seeds. 0.7 ml of solution is applied to 3.2 g of seeds 260 seeds). The seeds provided with a tacky coating are then shaken together with the ascospore powder, sieved and dried on the sieve. Each seed is coated with 150,000 to na, 600,000 spores. Measurement is made by suspending 10 seeds in 10 ml 0 a of water and determining from the suspension the number of spores o a Son contained therein with a hematometer.

o oQ 0o Po o 10 Example 3:

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0 o° Coating cotton seeds with a layer of ascospores of Chaetomium 0 000 globosum 0 0O o 10 g of cotton seeds 110 seeds) are sprayed in a fluidised bed n, n drier with 0.5 ml of a 0.6 solution of methyl cellosolve and the still tacky seeds are then rolled in a petri dish over an ascospore powder and then dried on a sieve.

0 0 0 Example 4: Coating sugar beet seeds with a mycelium of Chaetomium globosum A 250 ml Erlenmeyer flask is charged with 150 ml of a 2 malt extract (Oxoid) and this nutrient solution is inoculated with 1 ml of a spore suspension containing 105 ascospores per ml. The culture is incubated for 4 days at 24CC.

Then 10 ml of the oily culture broth is removed and 1.6 g of sugar beet seeds are added thereto. Seeds and broth are mixed for minutes at room temperature and the seeds are then allowed to drip on a sieve. The treated seeds are sown while still moist.

/1 K -9- Example Coating sugar beet seeds with iron phosphate and ascospores of Chaetomium globosum 3.2 g of sugar beet seeds are stirred at room temperature for minutes in a filtered solution of 1.36 g of iron phosphate in ml of a 0.36 molar solution of oxalic acid of pH 5.4. The seeds are poured onto a sieve and dried in a stream of air. After they have been dried, the seeds are coated with ascospores as described in Example 2.

jO Example 6 Coating seeds with chemical fungicide 0 0 0 6.4 g of seeds or 10 g of cotton seeds are stirred for 20 minutes at 0 .4 room temperature in 20 ml of a 0.1 solution of 3a,4,7,7a-tetrahydro-2-[(trichloromethyl)thio]-H-isoin e-,3(2H ne (Caan S o prepared by dissolving in 0.2 Orthozid 50 Sigfrie The seeds are then collected on a sieve and dried in a stream of air.

Example 7: Germination assays with ascospores of Chaetomium globosum The germination capacity of stored spores encapsulated in seed coats )O was compared with that of freshly harvested spores.

Spores which have been freshly harvested from cultures and samples of spores that have been stored for 1 to 15 months at 100-30°C and "i 20-60 relative humidity are placed on agar plates and examined in y climatic chambers at 22 0 C for their germination capacity.

Almost all spores examined germinated within 7 hours, in time to be able to protect the germinating seeds.

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l 99 9 o o 9 *o 9 9 9 9 10 Temperature Germination capacity after storage for during no storage storage 0 C) (fresh spores) 1 month 5 months 10 months 15 months 100 84 85 90 90 150 78 80 59 13 200 50 81 91 86 92 250 78 90 89 91 76 85 82 85 )1 Example 8: Assay for germination capacity of coated seeds Untreated as well as chemically treated sugar beet and cotton seeds are put into petri dishes (10 seeds per dish) on a thick layer of moist filter paper. The petri dishes are then put into a climatic chamber and kept under observation. The criterion for germination is the formation of roots.

The sugar beet seeds are kept for 12 hours in daylight at 20°C and 12 hours in the dark at 14°C and at 80-90 relative humidity.

Germination of the untreated seeds is 75 between the 3rd and 8th >o day and up to 90 on the 20th day. The treated seeds begin to germinate later after the 6th day and likewise achieve up to 85 germination by the 20th day.

The cotton seeds are kept for 14 hours in daylight at 25 0 C and hours in the dark at 200C. Germination of the untreated seeds is almost 100 between the 2nd and 8th day. Germination of the treated seeds is more than 90 Example 9: Assay for the protective action of sugar beet seeds coated with ascospores of Chaetomium globosum in a climatic chamber Untreated sugar beet seeds and seeds coated with ascospores as described in Example 2 are put into plastic dishes measuring x 12 x 4.5 cm and filled with soil. 20 seeds are sown in each I i dish, with each seed being sown individually into a 9 mm cavity made in the soil. The soil is disinfected (100'C) soil and soil which has been inoculated with Pythium ultimum and Rhizoctonia solani. The seed dishes are then kept in climatic chambers for 12 hours in daylight at 20C and 12 hours in the dark at 1400 and 80-90 relative humidity. The seed dishes filled with infected soil are prepared in the climatic chamber 3 days before sowing. Five dishes each containing 100 seeds are used per assay. The dishes are kept under daily observation and the perished plants are removed and used for mycological investigation of the pathogens.

The assay is terminated 3 weeks before sowing and the number and condition of the plantlets is determined.

The results are as follows: Surviving plants Seeds Soil inoculated with disinfected Pvthium Rhizoctonia

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t Sj ultimum solani untreated 79 18 7 treated with Chaetomium globosum 1.5.105 spores 63 68 according to Example 2 treated with 0.1 of Captan 88 53 according to Example 6 S 4555 V's's, S V S S I S S 4 5 Example Assay for determining the protective action of cotton seeds coated with ascospores of Chaetomium globosum in a climatic chamber Plastic dishes measuring 10 x 12 x 4.5 cm are filled with soil inoculated with Pythium ultimum and Rhizoctonia solani and kept for 3 days in a climatic chamber at 80-90 relative humidity for 16 hours in daylight at 260C and 8 hours in the dark at 200C. Then 9 cotton seeds are sown in each dish in a 15 mm cavity made in the V 4 12 soil. 10 dishes or 90 seeds are used for each assay. The dishes are kept under regular observation and the perished plants are removed and used for mycological investigation of the pathogens. The assay is terminated after 3 weeks and the number and condition of the germinated plantlets (in is determined.

Surviving plants Seeds Soil inoculated with disinfected Pythium Rhizoctonia ultimum solani untreated 76 16 20 treated with Chaetomium globosum 1.5-10 5 spores 76 58 according to Example 2 treated with 0.1 of Captan 84 40 according to Example 6 S' Example 11: Protective action of sugar beets provided with different protective coatings Sugar beet seeds are coated according to the method of Example 2 0C with different adhesive solutions and coated with ascospore powder.

The spore density was only 1.5*105 spores per seed. The seeds are compared with untreated seeds and with seeds treated with Captan (cf. Example 6).

Seed dishes are filled with soil which is naturally infected with Pythium ultimum. Then 20 sugar beet seeds are sown in each seed dish to a depth of 9 mm and the dishes are left in the climatic chamber under the same conditions and with regular watering. The germination of the seeds is monitored and perished plants are removed. The activity of the protective coating is evaluated 28 days after sowing by counting the number of surviving plants.

t I 13 Surviving plants in disinfected soil Seed coating soil infected with Pythium ultimum none 91 51 methyl cellulose 1.5-105 spores 1 methyl cellulose 1.5-105 spores xanthane 0.3 1.5-105 spores xanthane 1 1.5-105 spores 72 75 65 73 66 9 9 gum arabic 13 1.5-105 spores gum arabic 30 1.5-105 sp,res 77 none mycelium culture (Example 4) AO Captan, 0. 1 57 86 f C 9 t t t V t I 1 The same assay was carried out with soil inoculated with Pythium ultimum. The seeds were untreated, coated with 1 5 1 0 5 ascospores of Ohaetomiunt globosum according to Example 2, with iron phosphate and ascospores according to Example 5, or with Captan according to Example 6.

Iii-UI 14 Surviving Plants Seed coating disinfected solid inoculated with soil Pythium ultimum none 86% 9% methyl cellulose -48% 1.5105 ascospores tr iron phosphate 0.5% -67% meth.cell. 1.5.105 ascospores *t r Captan 0.1% -88% t r o tr Example 12: Field test Sugar beet seeds were sown in April with a mechanical sower in fields in CH-1374 Corcelles and CH-1373 Charvorney in Switzerland. The plants were counted after 36 days and evaluated. Each seed coating was treated in 6 test plots of one row of 12 m.

The plots were biologically examined and the following pathogenic microorganism were observed: Aphanomycetes cochlioides, Rhizoctonia sp., Fusarium Pythium debarvnum.

Untreated seeds and seeds coated with spores of Chaetomium ultimum according to Example 2 or seeds coated industrially with Thiram* (a registered trade mark) at (TMTD) were used for the test. The surviving plants were counted after 35 days.

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Seed coating Surviving plants Chavorney Corce lies none 42 according to Example 2 with 64 61% 1.5*1O5 ascospores industrially without fungicide 32 37 industrially with TMTD* 41 43 *Thiram or TMTD, tetramethylthiuram disulfide (CH3) 2

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3 2 IG Bayer. The treated seeds were obtained from Kleinwanzlebener Saatzucht in Einbeck, Germany.

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Claims (16)

1. A method of protecting emerging sugar beet and cotton plants against damping-o'" gases caused by soil-borne botanical microorganisms, which comprir.!s provLi,,g seeds of said plants with a protective coating which contains an effective amount of a culture extract of Chaetomium globosum.

2. A method according to claim 1 of protecting emerging sugar beet and cotton plants from damping-off diseases caused by soil-borne microorganisms, which comprises providing the seeds of said plants with a protective coating which contains ascospores of Chaetomium globosum.

3. A method according to claim 1 of protecting sugar beet plantlets from fungal damping-off diseases, which comprises providing sugar beet seeds with a protective coating which contain ascospores of Chaetomium globosum.

4. A method according to claim 1 of protecting cotton plantlets from fungal damping-off diseases,'which comprises treating cotton seeds with a protective coating which contain ascospores of Chaetomium globosum.

A method according co claim 1, wherein the protective coating contains an effective amount of 104-6 x 105 ascospores.

6. Seeds of sugar beet and cotton which are provided with a protective coating to protect against damping-off diseases caused by soil-borne botanical microorganisms which coating contains an effective amount of ascospores of Chaetomium globosum.

7. Sugar beet seeds which are provided with a protective coating which contains an effective amount of ascospores of Chaetomium globosum according to claim 6.

8. Cotton seeds which are provided with a protective coating which contains an effective amount of ascospores of Chaetomium globosum according to claim 6.

9. Seeds according to claim 6, the protective coating of which 4 5 contains 104-6 x 10 ascospores.

A method of providing seeds of useful plants with a coating to protect against damping-off diseases caused by soil-borne botanical microorganisms which coating contains ascospores of Chaetomium globosum, which method comprises: wetting the seeds with a liquid organic or inorganic solution which can be dried, 7y drying said seeds, 7Y 4 4 4 C tit t r 1- -r irN"- 2 (c) seeds with (d)

11. seeds. 17 L ore they are completely dry are are still tacky, mixing the a powder consisting of ascospores of Chaetomium globosum, and drying the coated seeds completely. A method according to claim 10, wherein the seeds are sugar beet r r I r 'rII *1 r *r I a Ii. ot I 0904 Ce., a C. I I C C I

12. A method according to claim 10, wherein the seeds are cottong seeds.

13. A method according to claim 10, wherein the solution which can be dried contains water, methyl cellulose and gum arabic.

14. A process according to claim 10, wherein the solution which can be dried contains water and iron phosphate.

15. A method according to claim 10, wherein the solution which can be dried contains water, clay particles, alginates and other formulation substances.

16. Seeds of sugar beet and cotton which are provided with a protective coating which contains an effective amount of ascospores of Chaetomium globosum, substantially as hereinbefore described with reference to any one of Examples 1 to 6. DATED this TWENTY-FOURTH day of JULY 1989 Ciba-Geigy AG Patent Attorneys for the Applicant SPRUSON FERGUSON 2s ~MR/7y 0PL14 'q