CA2107098A1

CA2107098A1 - Antifungal micro-organism

Info

Publication number: CA2107098A1
Application number: CA002107098A
Authority: CA
Inventors: Annabel Renwick; Keith A. Powell; Richard Campbell
Original assignee: Individual
Current assignee: Syngenta Ltd
Priority date: 1991-04-11
Filing date: 1992-04-03
Publication date: 1992-10-12
Also published as: HU9302694D0; WO1992018613A1; GB9107678D0; AU656369B2; BG98144A; AU1531592A; RU2104645C1; EP0579635A1; JPH06506345A; CZ280653B6; NZ242291A; BG61654B1; NO933623D0; FI934441A0; FI934441A; CZ210693A3; NO933623L; HUT67770A

Abstract

2107098 9218613 PCTABS00017 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

WO 92/18C13 1 PCI~/GB92/QOSg7 ~ 21~7~8 ANTIFUNGAr, MICRO--ORGANISM

This invention relates to an agricultural ; plant protection product. More specifically, the invention relate$ to an antifungal micro-organ$~m, in partlcular to a novel strain of Pseudomonts fluorescens, and the uses thereof for the protection of plants against fungal attack.
Certa~n 6trains of the micro-organism Pseudomonas fluorescens are known to be useful agricultural fungicides. The antifungal activity varies greatly amongst the innumerable strains of the micro-organism. Agriculturally useful strains are those which posse6s useful levels of activity against specific target fungi. One such target is the group of fungi which are associated with the disease known as ~damping-off~ which is partlcularly troublesome in certa$n crops and under particular climatic conditions. U80 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 ob~ect of the present invention i8 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.
~he invention also comprises an antifungal agricultural composition containing Pseudomonas fluQrescens Biovar r, strain NCIB 40189 as active ingredient in admixture with a carrier composit1on WO~lK13 2 21 0 7 ~ 9 8 PCT~GB9~g7 acceptable in agricultural practice. Exam~les of the types of agricultural formulation~ which may be employed are seed coatings compositions, liguid 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 seedg thereof or the growing medium for the plant a fungicidally effective dosage of the said Pseudomonas fluorescens Biovar I, gtrain NCIB
40189. The treatment may be effected by application of a preparation containing organismg of the strain of this invention.
The antifungal micro-organism of this invention is particularly effective against fungal infections asgociated with ~damping-off~ disease, guch as fungi of the genera Rhizoctonia, Pythium, Fusarium. It is also active against .
Gaeumannomvces ~raminis which causes take-all digease .
The strain of this invention (NCIB 40189) was isolated from volunteer wheat roots collected from plants grown at Chiseldon, Swindon, Wiltshire, United Ringdom. It was deposited at The National Collections of Industrial and Marine Bacteria ~23 St Machar Drive, Aberdeen, Scotland, AB2 lRY) 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.

WO g2/18613 3 PC~GB~2/00597 ISOLATION OF Tlll~ MICRO--ORGANISM
The bacterium was isolated from volunteer wheat root~ dug from a field at a farm known a~
Draycott Farm, Chiseldon, Swindon, Wiltshire, United Kingdom.
Th~ root6 were washed to remo~e all adhering soil and placed in sterile distilled water in a flask containing glass beads. The roots were shaken with the glas~ beads for 15 minutes and the wa~hing& then plated out on to Ring~ B agar ~Proteose Peptone No.3, 20g; glycerol lOml~
pota8sium sulphate, 1.5g; maqnesium sulphate heptahydrate, 1.5g; technical agar, 12gS and, one lltre of d~stilled water at p~ 7.2 and $ncludlng cycloheximide 75 micrograms/ml5 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 20C.
The bacterium was taken at random from the agar plate and ~ub-cultured on to 1/10 trypt~c soya broth agar (TSA) (3g tryptic 80ya broth; 15g agar; one l~tre water). The bacterium was maintained an 1/10 TSA and 6tored at 4C until required.
C~ARACTERISATION OF TEIB MICRO--ORGANISM
The morphological characteri6tlcs of Pseudomonas fluorescens strain NCIB 40189 were determined by the National Collection of Industrial and Marine ~acteria and are #hown in Table 1 below.

, , ' - ' .
- ~ ~

21~ 7 ~ 9 8 PCT/GB~K97 Morphology of P iluore wens strain NCIB 40189 Cell Morphology: Gram negative rods Fluorescence: +ve - RAPID TEST (~P~) - Nitrate reduction Indole production Acid from glucose (~) Arginine dehydrolase +
ureage Aesculin hydrolysis Gelatin hydrolysis +
~-Galactos~dase Glucose ass~milation +
Arabinose assimilation +
Mannose assimilation +
Nannitol assimilation +
N-acetylglucosamine assimilation +
Naltose 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 : WO ~VIK13 5 PCT/GB~00597 ~`- 21~7~98 Residual nitrogen +
DNAa~e CSU:--Acetate +
Trehalose +
meso-Inositol Adonitol Butyrate Propylene glycol Gel Stab 20C
Gel Plate +
Casein (+) Starch Leclth egg ~ipase egg H2S ~TSI) Tween 80 +
Vrease +
Phenylalanine deaminase Growth factor required Peniclllln G
8treptomycln +
Chloramphenicol +
Tetracycl~ne +
Novoblocin Polymyxin B +
~evan +

~Tests are described in: R E Buchanan, N E Gibbons 30 (eds), 1974, Bergey's Manual of Determinatlve Bacteriology, Bth ed; S T Cowan, R J Steel, 1974, Manual for Identification of Medical 8acteria).

WO92/18613 6 210 7 0 9 8P~/GB92/00S97 PRII~ARY SOIL PEq'RI--DISEI SCRI~N
Pseudomonas fluorescens strain ~CIB 40189 was tested for activity against Pythium ultimum, using a petri-dish soil screen.
S Petri-dlshes (5cm) were filled with 10ml of potato dextrose agar (PDA). Agar plugs (5mm) from a 7 day old culture of Pythium ultimum grown at 20C were placed centrally on to the PDA. The pl~te~ were incubated for 5 days at 20C during which the P ultimum colonised the plate. Minster Mendip ~oam was autoclaved at 120C for 60 minutes. To this sterile wheatgerm was added (1%
w/w) and mixed well. Approximately 8g of ~oil 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 (TS8J for 48 h at 12 C);

2) M~TAIAXYL (100 ppm AI);

3) CONTRO~: 1/10 TSB alone.
Duplicate plates were prepared, and lncubated for 4 days at 10C.
Results are shown in Table 2 below. Actlvity 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 wa~ no mycelial growth in the dish.

WO~UI8613 7 ~CT/~B9~97 ~ 1 ~ 7 8 9 8 Activity of NCIB 40189 against P ulti~u~

TREATMENT ACTIVITY SCORE

NCIB 40189 . 3 Metalaxyl 3 Control O

The results show that strain NCIB 40189 inhibited fungal growth in the soil. Its activity is comparable to Metalaxyl, a standard chemical fungicide.

S~CONDARY POT SCR~FNING
.
~he effectiveness of Pseudomonas fluorescens strain NCIB 40189 was further tested against Pythium ultimum and Rhizoctonia solani. Results show that strain NCSB 40189 has activity against P
ultimum and R solan~ under glasshouse conditions.

ACTIVITY AGAINST P U~TIMUM:
Cultures of P ultimum were raised on PDA
plates for 7 days at 20C. A mixture of 200g of silver sand, Sg cornmeal, 4g Bemax wheatgerm and 40ml water wAs autoclaved at 120C for 20 minutes then placed in a flask and inoculated with 1/4 of a plate of Pythium. The flasks were incubated at 20C for 7 days, then the contents of each flask wa~ 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 :, :

, . .

~VO~VIK13 ~ 7~98 Mendip loam to give a dilut~on 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 80il.
The following treatments were used (35 ml added as a drench to each pot):
1) Pseudomonas fluorescens strain NCIB 40189 (culture grown $n 1/10 tryptic soya broth (TSB) for 4~ h at 20C);
2) METAhAXYL (0.1-5.0 ppm AI);
3) CONTROL: 1/10 TSB alone.
Five ~eplicates were prepared for each treatment.
Pea plants were grown for 14 days at 15C, and seedling emergence was monitored weekly. The plants were watered da$1y: particular care was taken to ensure that the soil was kept moist during the tests as the disease inc$tes damping-off under cool, damp conditions.
Results are shown in Table 3 below.

.' '' ' " ' ~- . '.:' - .

WO 9V18C13 9 PCl'~GB92/00597 ,-- 2107~98 Effect of strain NCI8 40189 on e~tablish~ent of pea~ ln the presence of P ultimum TREATMENT Percentage ~%) emergence of peas in pots wlth P ultimum present Metalaxyl 0.1 ppm 64 0.5 ppm 84 1.0 ppm 84 5.0 pp~ 96 Control 28 Result~ for strain NCIB 40189 and for the untreated control show that application of the Pseudomonas fluorescens strain allows slgnificantly more pea plants to emerge. Act~vlty of ~train NCIB 40189 is comparable wlth that of the chemical ~ungicide Metalaxyl at tho disoa~e levels expressed.

ACTIVITY AGAINST R SOLANI:
Cultures of R solani were raised on PDA plates for 7 days at 20C. A mixture of 200g of silver ~and, 4g Bemax wheatgerm and 40ml water was autoclaved at 120C for 20 minutes then placed in a fla~k and inoculated with l/4 of a plate of Rhizoctonla. ~he flasks were incubated at 20C
for 7 days, then the contents of each flask was . .
' WO9~18613 l0 2 1 ~ 7 ~ 9 ~CT/GB9~97 mixed with fine sand to a final weight o~ 300g.
Three quarters of a flask of Rhizoctonia inoculum was mixed with 6 litres of Mendip Minster ~oam to give a standard dilution (Y). This was further diluted with clean Mendip loam to give a dilution of Y/B used in the tests.
Tests were carried out in pots (3 inches diametsr), 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):
l) Pseudomonas fluorescens strain NcrB 401~9 (culture grown in l/10 tryptic soya broth (TSB) for 48 h at 20C);
2~ PENCYCURON (Monceren at l0 or l00 ppm AI);
3) DISEASED CONTRO~: l/l0 TSB alone;

4) HEALTHY CONTROL: l/l0 TSB alone on soil not infested with Rhizoctonia.
Five replicates were prepared for each treatment.
Pea plants were grown for 14 days at 24C, and seedling emergence was monitored weekly. The plants were watered daily.
Results are shown in Table 4 below.

' . ' :. . ' .,, .

.

~ ~ ' : - ' ' wo ~UI8613 11 210 7 0 9 8 PCT/GB9~97 Effect of ~train NCIB 40189 on establish~ent of peas in the presence of R solani TREATMENT Percentage.(%) emergence of peas Monceren 1010 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 campared to the d~seas~d control. ~n fact, application of strain NCIB
40189 allows pea establishment at a level comparable to the hoalthy control ~treatment wlthout R solani). Activity of the BCA also appears to bo higher than that of the chomical fungicide Monceren.

PROTECTION AGAINST Pr,ANT D~SEASE: FVRT~IER TESTS
Pseudomonas fluorescens strain NCIB 40189 was further tested for its activity against Rhizactonia solani and its abillty to protect pea plants from disease.
Tho trial was carried out in rectangular trays ~165mm x 95mm), half-filled with infested soil (prepared according to the method described in the . ' . ! .
- - ' :- :
. .

WO ~VI86I3 12 2i~7~9~ PCT/GB~U~97 previous section). Twenty-five peas were sown in each tray and covered with clean soil.
The B~A (strain NCIB 40189) and Control treatments were as follows:
1) DRENC~ 100 ml bacterial suspension (109 cells/ml) added to each tray;
2) GRANU~ES lO g pre-formed granules added to each tray;
granules (types A, ~, C) carrying bacterial cells were inoculated at a maximum capacity of BCA
concentrate: 4.5x101 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 24C. 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.

- .
;;
, ~ . .

WO~U18613 13 PCT/GB~OOSs7 210709~
T~LE 5 Activity of strain NCIB 40189 again~t R solanl TREATMENT Mean percentage emergence (%) of pea~
s from R solani infested compost Drench 73 Granule A 65 Granule ~ ~4 Granule C 66 Untreated 31 Application of stra$n NCI~ 40189 significantly $ncreased the percentage emergence of the plants when compared to the untreated control. ~here is no statistically significant difference between re~ults obtained with the drench or granule BCA treatments.

FIELD TRIA~S
Pseudomonas fluorescens straln NCIB 40189 Wa8 used ln field trlals as a biological control agent (BCA) again~t ~damping-off~ d~sease ~n peas and maize. Field trials of strain NC~B 40189 have boen conducted on its potential for inhibltlng Pythlum and Fusarlum spp. Results were compared to standard chemical treatments, and are reported below. ~n 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 chemlcal treatment.

' WO~Ut8613 14 2 1 0 7 ~ 9 ~CT/GB~K97 ~. . .
.

EFFICACY AGAINST PYTEII~
Trials were conducted in France using peas as the test crop. Pathogen was added at various rates as whaat grain inoculum, prepared as follows. satches of wheat seed (1 Kg) were soaked in water overnight, drained and autoclaved at 121~C. Fungal spores which rema~ned viable were allowed to germinate and grow for two days before the 6eed was re-autoclaved at 121~C. A 7 day old plate of PYthium ultimum (grown on potato dextrose agar at 20C) 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 (107 cell/seed). APRON (Trade Nark) was used as a chemical control, applied at a rate of 30g AI/lOORg seed. Two types of plant control were used: untreated pea seed, and blank pea seed (which had undergone seed treatment without addit~on 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 2ao ceeds 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.

WO~U18613 15 2 1 0 7 G 9 8 PCT/GB9~K97 Field ~ctivity of NCIB 40189 again8t P ulti~u :
e~ergence of pea plants ;-TREATMENT NUMBE~ EMERGED WHEN P ULTIMUM
ADDED AT T~E FO~BOWING RATES(g inoculum per metre row):

NCIB 40189 180.2~ AB 153.00 CDG 134.50 FG~
APRON 171.50 AC 169.50 ACE 157.25 BCDF
Untreated 172.50 AC 130.25 GHI 111.25 I
alank 176.00 AC 144.34 DE~ 119.00 ~I

The fir6t 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 ~oil allowed normal plant growth. There i~ no real significant d~fference between treatments, indicatlng the addition of strain NCIB 40189 had no phytotoxic effect.
The other figures in Table 6 givo the results for two test treatments at different pathogen rates. The presence of pathogen in the 80il 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 affectlng plant growth.
~owever, the presence of strain NCIB 40189 ~ignificantly increases the number of plants emerging in the presence of the pathogen.

.

WO~U18613 16 21 0 7 o ~/GBg~g7 EFFICACY AGAINST FUSARIUM:
Field trials were carried out in the USA
(Mississippi), using maize as the test crop.
Pathogen (F solani) was added at various rates as wheat grain inoculum, prepared as descrlbed above.
Stra~n NCIB 40189 was applied as a gum/peat formulation (107 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 l5 days after sowing. The letters appearing after the percentage emerged indicate statistical s~gnificance. There is no significant dif~erence at the 5% probability level between entries w~th a letter in common.

TA~LE 7 Field ~ctlYity of NCIB 40189 again~t F solani:
emergence of maize plants T~EATMENT PERCENTAGE ~%) EMERGED W~EN F SOhANT
ADDED AT THE FOL~OWING RATES
(g inoculum per metre row):

NCIB 40189 65.208 C 74.583 AC
CAPTAN 69.583 BC 69.375 BC
Vntreated 65.833 C 51.250 D

The first column of Table 7 shows results for the control tria}s where no test pathogen was added. The absence of pathogen ~n the so~l W0~18613 17 21 0 7 0 9 8 allowed normal plant growth. There ~8 no real significant difference between treatments, indicatlng the addit$on of strain NCIB 40189 had no phytotoxic effect.
S The presence of pathogen, Fusarium solan~, in the soil decreased the number of plants emerging from untreated seed when compared to the control experlment. This shows disease was present and wa~ adversely affect~ng plant growth. ~owever, the presence of ~tra~n NCIB 40189 significantly increases the percentage of plants emerging in the presence of the pathogen. In fact, treatment wlth strain NCIB 40189 improved plant performance 80 that the percentage emerging was not significantly different from the percentage emerging in the absence of pathogen.

Claims

1. An antifungal agent comprising the micro-organism Pseudomonas fluorescens strain NCIB 40189, 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.