ANTIFUNGAL MICRO-ORGANISM

Abstract: A new strain of Pseudomonas fluorescens which displays antifungal activity on a wide spectrum of disease fungi has been isolated. Screening, testing and field trial results are provided. The antifungal agent is particularly active against "damping-off" disease caused by fungi of such genera as Rhizoctonia, Pythium, and Fusarium. Methods of using such micro-organisms and fungicidal compositions containing same to protect crop plants from fungal infection are described. The antifungal agent can be used as an agricultural fungicide, particularly as a seed coating.

Description: ANTIFUNGAL MICRO-ORGANISM This invention relates to an agricultural plant protection product. More specifically, the invention relates to an antifungal micro-organism, in particular to a novel strain of Pseudomonas fluorescens, and the uses thereof for the protection of plants against fungal attack. Certain strains of the micro-organism Pseudomonas fluorescens are known to be useful agricultural fungicides. The antifungal activity varies greatly amongst the innumerable strains of the micro-organism. Agriculturally useful strains are those which possess useful levels of activity against specific target fungi. One such target is the group of fungi which are associated with the disease known as "damping-off" which is particularly troublesome in certain crops and under particular climatic conditions. Use of Biological Control Agents (BCAs) or biopesticides to control disease may be more effective or more preferable to the use of standard chemical control agents. An object of the present invention is to provide an agriculturally useful fungicide. According to the present invention there is provided a novel strain of Pseudomonas fluorescens Biovar I, deposited at The National Collection of Industrial and Marine Bacteria under the Accession Number NCIB 40189 on 1st September 1989. The invention also comprises an antifungal agricultural composition containing Pseudomonas fluorescens Biovar I, strain NCIB 40189 as active ingredient in admixture with a carrier composition acceptable in agricultural practice. Examples of the types of agricultural formulations which may be employed are seed coatings compositions, liquid for root or soil drenching and granular or powder compositions. The base materials for these are well known in the art. Additionally, the invention provides a method of protecting crop plants from fungal infection comprising applying to the plants, the roots or seeds thereof or the growing medium for the plant a fungicidally effective dosage of the said Pseudomonas fluorescens Biovar I, strain NCIB 40189. The treatment may be effected by application of a preparation containing organisms of the strain of this invention. The antifungal micro-organism of this invention is particularly effective against fungal infections associated with "damping-off" disease, such as fungi of the genera Rhizoctonia, Pythium, Fusarium. It is also active against Gaeumannomyces graminis which causes take-all disease. The strain of this invention (NCIB 40189) was isolated from volunteer wheat roots collected from plants grown at Chiseldon, Swindon, Wiltshire, United Kingdom. It was deposited at The National Collections of Industrial and Marine Bacteria (23 St Machar Drive, Aberdeen, Scotland, AB2 1RY) under the terms of the Budapest Treaty on 1st September 1989. There now follows a description of the isolation, characterisation and screening of the bacterial isolate with examples of its activity against several fungi which infect crop plants. ISOLATION OF THE MICRO-ORGANISM The bacterium was isolated from volunteer wheat roots dug from a field at a farm known as Draycott Farm, Chiseldon, Swindon, Wiltshire, United Kingdom. The roots were washed to remove all adhering soil and placed in sterile distilled water in a flask containing glass beads. The roots were shaken with the glass beads for 15 minutes and the washings then plated out on to Kings B agar (Proteose Peptone No.3, 20g; glycerol 10ml; potassium sulphate, 1.5g; magnesium sulphate heptahydrate, 1.5g; technical agar, 12g; and, one litre of distilled water at pH 7.2 and including cycloheximide 75 micrograms/ml; chloromycetin 12.5 micrograms/ml; and ampicillin 50 micrograms/ml) at 10:1 and 10:3 dilutions. The plates were incubated for three days at 20 β C. The bacterium was taken at random from the agar plate and sub-cultured on to 1/10 tryptic soya broth agar (TSA) (3g tryptic soya broth; 15g agar; one litre water). The bacterium was maintained an 1/10 TSA and stored at 4°C until required. CHARACTERISATION OF THE MICRO-ORGANISM The morphological characteristics of Pseudomonas fluorescens strain NCIB 40189 were determined by the National Collection of Industrial and Marine Bacteria and are shown in Table 1 below. TABLE 1 Morphology of P fluorescens strain NCIB 40189 Cell Morphology: Gram negative rods Fluorescence: +ve RAPID TEST (APT) Nitrate reduction - Indole production - Acid from glucose (+) Arginine dehydrolase + Urease - Aesculin hydrolysis - Gelatin hydrolysis + β-Galactosidase Glucose assimilation + Arabinose assimilation + Mannose assimilation + Mannitol assimilation + N-acetylglucosamine assimilation + Maltose assimilation Gluconate assimilation + Caprate assimilation + Adipate assimilation - Malate assimilation + Citrate assimilation + Phenylacetate assimilation - Cytochrome oxidase + Pyocyanin — Fluorescence + Gas Glucose - Acid Glucose PNS + ONPG Nitrate to nitrite Nitrate to nitrogen - Residual nitrogen + DNAase CSU:- Acetate + Trehalose + meso-Inositol Adonitol Butyrate + Propylene glycol - Gel Stab 20 β C + Gel Plate + Casein (+) Starch Lecith egg - Lipase egg H 2 S (TSI) Tween 80 + Urease + Phenylalanine deaminase - Growth factor required Penicillin G Streptomycin + Chloramphenicol + Tetracycline + Novobiocin - Polymyxin B + Levan + (Tests are described in: R E Buchanan, N E Gibbons (eds), 1974, Bergey's Manual of Determinative Bacteriology, 8th ed; S T Cowan, K J Steel, 1974, Manual for Identification of Medical Bacteria). PRIMARY SOIL PETRI-D SH SCREEN Pseudomonas fluorescens strain NCIB 40189 was tested for activity against Pythium ultimum, using a petri-dish soil screen. Petri-dishes (5cm) were filled with 10ml of potato dextrose agar (PDA). Agar plugs (5mm) from a 7 day old culture of Pythium ultimum grown at 20°C were placed centrally on to the PDA. The plates were incubated for 5 days at 20°C during which the P_ ultimum colonised the plate. Minster Mendip Loam was autoclaved at 120°C for 60 minutes. To this sterile wheatgerm was added (1% w/w) and mixed well. Approximately 8g of soil was placed into each Petri dish to cover the Pythium lawn. The following treatments were used (2 ml applied per petri-dish) : 1) Pseudomonas fluorescens strain NCIB 40189 (culture grown in 1/10 tryptic soya broth (TSB) for 48 h at 12 °C); 2) METALAXYL (100 ppm Al) ; 3) CONTROL: 1/10 TSB alone. Duplicate plates were prepared, and incubated for 4 days at 10°C. Results are shown in Table 2 below. Activity was scored on a scale of zero to 3. A score of zero meant there was considerable growth of Pythium; a score of 3 meant there was no mycelial growth in the dish. TABLE 2 Activity of NCIB 40189 against P ultimum TREATMENT ACTIVITY SCORE NCIB 40189 3 Metalaxyl 3 Control 0 The results show that strain NCIB 40189 inhibited fungal growth in the soil. Its activity is comparable to Metalaxyl, a standard chemical fungicide. SECONDARY POT SCREENING The effectiveness of Pseudomonas fluorescens strain NCIB 40189 was further tested against Pythium ultimum and Rhizoctonia solani. Results show that strain NCIB 40189 has activity against P ultimum and R solani under glasshouse conditions. ACTIVITY AGAINST P ULTIMUM: Cultures of P ultimum were raised on PDA plates for 7 days at 20°C. A mixture of 200g of silver sand, 5g cornmeal, 4g Bemax wheatger and 40ml water was autoclaved at 120°C for 20 minutes then placed in a flask and inoculated with 1/4 of a plate of Pythium. The flasks were incubated at 20°C for 7 days, then the contents of each flask was mixed with fine sand to a final weight of 300g. One flask of Pythium was mixed with 8 litres of Mendip Minster Loam to give a standard dilution (X). This was further diluted with clean Mendip loam to give a dilution of X/16 used in the tests. Tests were carried out in pots (3 inches diameter), filled to 3/4 with infested soil. Five peas were sown in each pot and covered with clean soil. The following treatments were used (35 ml added as a drench to each pot): 1) Pseudomonas fluorescens strain NCIB 40189 (culture grown in 1/10 tryptic soya broth (TSB) for 48 h at 20°C); 2) METALAXYL (0.1-5.0 ppm Al) ; 3) CONTROL: 1/10 TSB alone. Five replicates were prepared for each treatment. Pea plants were grown for 14 days at 15°C, and seedling emergence was monitored weekly. The plants were watered daily: particular care was taken to ensure that the soil was kept moist during the tests as the disease incites damping-off under cool, damp conditions. Results are shown in Table 3 below. TABLE 3 Effect of strain NCIB 40189 on establishment of peas in the presence of P ultimum TREATMENT Percentage (%) emergence of peas in pots with P ultimum present NCIB 40189 76 Metalaxyl 0.1 pp 64 0.5 ppm 84 1.0 ppm 84 5.0 ppm 96 Control 28 Results for strain NCIB 40189 and for the untreated control show that application of the Pseudomonas fluorescens strain allows significantly more pea plants to emerge. Activity of strain NCIB 40189 is comparable with that of the chemical fungicide Metalaxyl at the disease levels expressed. ACTIVITY AGAINST R SOLANI: Cultures of R solani were raised on PDA plates for 7 days at 20°C. A mixture of 200g of silver sand, 4g Bemax wheatgerm and 40ml water was autoclaved at 120°C for 20 minutes then placed in a flask and inoculated with 1/4 of a plate of Rhizoctonia. The flasks were incubated at 20°C for 7 days, then the contents of each flask was mixed with fine sand to a final weight of 300g. Three quarters of a flask of Rhizoctonia inoculum was mixed with 6 litres of Mendip Minster Loam to give a standard dilution (Y). This was further diluted with clean Mendip loam to give a dilution of Y/8 used in the tests. Tests were carried out in pots (3 inches diameter), filled to 3/4 with infested soil. Five peas were sown in each pot and covered with clean soil. The following treatments were used (35 ml added as a drench to each pot): 1) Pseudomonas fluorescens strain NCIB 40189 (culture grown in 1/10 tryptic soya broth (TSB) for 48 h at 20°C); 2) PENCYCURON (Monceren at 10 or 100 ppm Al); 3) DISEASED CONTROL: 1/10 TSB alone; 4) HEALTHY CONTROL: 1/10 TSB alone on soil not infested with Rhizoctonia. Five replicates were prepared for each treatment. Pea plants were grown for 14 days at 24°C, and seedling emergence was monitored weekly. The plants were watered daily. Results are shown in Table 4 below. TABLE 4 Effect of strain NCIB 40189 on establishment of peas in the presence of R solani TREATMENT Percentage (%) emergence of peas NCIB 40189 88 Monceren 10 ppm 40 100 ppm 60 Diseased control 20 Healthy control 92 Application of the Pseudomonas fluorescens strain NCIB 40189 allows significantly more pea plants to emerge when compared to the diseased control. In fact, application of strain NCIB 40189 allows pea establishment at a level comparable to the healthy control (treatment without R solani) . Activity of the BCA also appears to be higher than that of the chemical fungicide Monceren. PROTECTION AGAINST PLANT DISEASE: FURTHER TESTS Pseudomonas fluorescens strain NCIB 40189 was further tested for its activity against Rhizoctonia solani and its ability to protect pea plants from disease. The trial was carried out in rectangular trays (165mm x 95mm), half-filled with infested soil (prepared according to the method described in the previous section). Twenty-five peas were sown in each tray and covered with clean soil. The BCA (strain NCIB 40189) and Control treatments were as follows: q 1) DRENCH 100 ml bacterial suspension (10 cells/ml) added to each tray; 2) GRANULES 10 g pre-formed granules added to each tray; granules (types A, B, C) carrying bacterial cells were inoculated at a maximum capacity of BCA concentrate: 4.5x10 cells/g; 3) UNTREATED 100 ml water added as a drench to each tray as a control. Three replicates were prepared for each treatment. Pea plants were grown for 7 to 14 days at 24°C. The plants were watered daily and seedling emergence was monitored weekly. Test results were collated from all experiments and are shown in Table 5 below. TABLE 5 Activity of strain NCIB 40189 against R solani Application of strain NCIB 40189 significantly increased the percentage emergence of the plants when compared to the untreated control. There is no statistically significant difference between results obtained with the drench or granule BCA treatments. FIELD TRIALS Pseudomonas fluorescens strain NCIB 40189 was used in field trials as a biological control agent (BCA) against "damping-off" disease in peas and maize. Field trials of strain NCIB 40189 have been conducted on its potential for inhibiting Pythium and Fusarium spp. Results were compared to standard chemical treatments, and are reported below. In summary, these results show that disease caused by Pythium ultimum or Fusarium solani is reduced by application of strain NCIB 40189 to a level comparable with that obtained by standard chemical treatment. EFFICACY AGAINST PYTHIUM: Trials were conducted in France using peas as the test crop. Pathogen was added at various rates as wheat grain inoculum, prepared as follows. Batches of wheat seed (1 Kg) were soaked in water overnight, drained and autoclaved at 121°C. Fungal spores which remained viable were allowed to germinate and grow for two days before the seed was re-autoclaved at 121°C. A 7 day old plate of Pythium ultimum (grown on potato dextrose agar at 20°C) was added to the wheat seed and allowed to grow for 10 days, shaken to redistribute the mycelia, and left for a further 11 day growth period. The inoculum was then ready for use within the next 14 days. Strain NCIB 40189 was applied as a gum/peat formulation (10 cell/seed). APRON (Trade Mark) was used as a chemical control, applied at a rate of 30g AI/lOOKg seed. Two types of plant control were used: untreated pea seed, and blank pea seed (which had undergone seed treatment without addition of BCA or chemical). Results are shown in Table 6 below. "Number emerged" refers to the mean number of plants which emerged 22 days after sowing from 200 seeds on a strip 5 metres long. The letters appearing after the numbers "emerged" indicate statistical significance: there is no significant difference at the 5% probability level between entries with a letter in common. TABLE 6 Field activity of NCIB 40189 against P ultimum: emergence of pea plants TREATMENT NUMBER EMERGED WHEN P ULTIMUM ADDED AT THE FOLLOWING RATES (g inoculum per metre row): 0 100 200 NCIB 40189 180.25 AB 153.00 CDG 134.50 FGH APRON 171.50 AC 169.50 ACE 157.25 BCDF Untreated 172.50 AC 130.25 GHI 111.25 I Blank 176.00 AC 144.34 DEH 119.00 HI The first column in Table 6 shows results for the control experiment where tests were conducted in normal field soil, without any added pathogen. The absence of pathogen in the soil allowed normal plant growth. There is no real significant difference between treatments, indicating the addition of strain NCIB 40189 had no phytotoxic effect. The other figures in Table 6 give the results for two test treatments at different pathogen rates. The presence of pathogen in the soil decreased the number of plants emerging from untreated or blank seed when compared to the control experiment. This shows disease was present and was adversely affecting plant growth. However, the presence of strain NCIB 40189 significantly increases the number of plants emerging in the presence of the pathogen. EFFICACY AGAINST FUSARIUM: Field trials were carried out in the USA (Mississippi), using maize as the test crop. Pathogen ( _ solani) was added at various rates as wheat grain inoculum, prepared as described above. Strain NCIB 40189 was applied as a gum/peat formulation (10 cell/seed). The chemical control was CAPTAN (Trade Mark), used as a seed treatment at the recommended rate. Untreated seed was used as the plant control. Results are shown in Table 7 below. Emergence is quoted as percentage (%) of plants 15 days after sowing. The letters appearing after the percentage emerged indicate statistical significance. There is no significant difference at the 5% probability level between entries with a letter in common. TABLE 7 Field activity of NCIB 40189 against F solani: emergence of maize plants TREATMENT PERCENTAGE (%) EMERGED WHEN F_ SOLANI ADDED AT THE FOLLOWING RATES (g inoculum per metre row): 0 10 NCIB 40189 65.208 C 74.583 AC CAPTAN 69.583 BC 69.375 BC Untreated 65.833 C 51.250 D The first column of Table 7 shows results for the control trials where no test pathogen was added. The absence of pathogen in the soil allowed normal plant growth. There is no real significant difference between treatments, indicating the addition of strain NCIB 40189 had no phytotoxic effect. The presence of pathogen, Fusarium solani, in the soil decreased the number of plants emerging from untreated seed when compared to the control experiment. This shows disease was present and was adversely affecting plant growth. However, the presence of strain NCIB 40189 significantly increases the percentage of plants emerging in the presence of the pathogen. In fact, treatment with strain NCIB 40189 improved plant performance so that the percentage emerging was not significantly different from the percentage emerging in the absence of pathogen.

Claims: CLAIMS An antifungal agent comprising the micro-organism Pseudomonas fluorescens, a culture of which was deposited on 1st September 1989 under the terms of the Budapest Treaty with the National Collection of Industrial and Marine Bacteria Limited, Aberdeen, United Kingdom, under the Accession Number 40189. 2. An antifungal agricultural composition containing as active ingredient an antifungal agent claimed in claim 1, in admixture with a carrier composition acceptable in agricultural practice. 3. A method of inhibiting fungal attack on a plant, comprising applying an effective dose of an antifungal agent as claimed in claim 1 to the locus of a plant or seed. 4. A method as claimed in claim 3 where the antifungal agent is applied to a plant. 5. A method as claimed in claim 3 where the antifungal agent is applied to a seed prior to sowing. 6. A method as claimed in claim 3 where the antifungal agent is applied to a growing medium in which a plant is growing or is to be grown. 7. A method as claimed in claim 3 where the fungus is from the genus Rhizoctonia. 8. A method as claimed in claim 3 where the fungus is from the genus Pythium. 9. A method as claimed in claim 3 where the fungus is from the genus Fusarium. 10. A method as claimed in claim 3 where the fungus is Gaeumannomyces graminis.

Inventors: CAMPBELL RICHARD, POWELL KEITH ADRIAN, RENWICK ANNABEL, Campbell, Richard, POWELL, KEITH, ADRIAN, RENWICK, ANNABEL
Applicants: ICI PLC, IMPERIAL CHEMICAL INDUSTRIES PLC
Application year: 1992
Main classification: C12R1
IPC codes: A01N63, C12N1, C12R1