GB2036792A - Compounds Derived from a Bacterium and Having Antagonistic Activity - Google Patents

Abstract

The invention provides a chemical compound or a mixture of at least two chemical compounds separated from the bacterium P. antimicrobica and having fungistatic or bacteriostatic activity. The chemical compound or the mixture may be incorporated into a composition comprising, in addition to the compound or mixture, an inert diluent or carrier. It is envisaged that P. antimicrobica and the compound or mixture separated therefrom will be useful for combatting fungal and bacterial infections of animals, humans and plants and for suppressing or controlling fungal and bacterial growth in or on substrates. Cultures of the bacterium deposited in the culture collection of the National Collection of Industrial Bacteria have been given the accession numbers 9897 and 9898.

Description

SPECIFICATION Compounds Derived from a Bacterium and Having Antagonistic Activity Field of the Invention The present invention relates to a bacterium, which I have discovered exhibits antagonistic activity, to the use of the bacterium to exploit its antagonistic activity, to chemical compounds separated from the bacterium and having antagonistic activity and to the use of such compounds to exploit their antagonistic activity.

Description of the Invention I originally isolated the bacterium from an insect virus vector, and I have named the bacterium Pseudomonas antimicrobica.

Two cultures of P. antimicrobica were deposited in the culture collection of the National Collection of Industrial Bacteria on 20th July 1966, and were given the accession numbers 9897 and 9898 respectively.

P. antimicrobica has the following characteristics: Cells are straight, non-sporeforming, coccoid rods, 0.5 ,nx0.8-1 .5 a occurring singly, in groups and occasionally in pairs; Gram-negative, aerobic, heterotrophic and motile with 1-2 polar flagella.

Five-day old colonies on nutrient agar are circular, raised creamy white and opaque, varying from rugose and butyrous with raised micro-undulate margins (variant NCIB 9897) to moist smooth, glistening and slightly viscid with entire margins and occasionally with an orange tinge (variant NCIB 9898). Four-day-old nutrient broth cultures show turbidity, and thin to membranous pellicles which break easily when disturbed.

A yellowish-orange water-soluble, non-fluorescent pigment, which is readily diffusable, is produced, in media A and B of King, E.O., Ward, M.K. 8 Raney, D.E. (1954), J. Lab. clin. Med. 44,301. A weakly orange pigment is produced in Czapak Dox, Nutrient and many other common laboratory agar media, but none in Potato Dextrose agar.

Growth occurs from 1 50C to 37"C with a broad optimum temperature range of 30 to 370C; There is no growth at 41 OC. Growth (NCIB 9898) also occurs in the presence of 0.5%, 2% and 4% NaCI, with an optimum at 24%, but weakens to a trace at 6%.

Acid but not gas is produced from glucose, lactose, fructose, xylose, galactose, arabinose, manitol, dulcitol, sorbitol, glycerol and meso-inositol. There is no acid or gas production from salicin, maltose, sucrose and raffinose. Starch and ethanol are not attacked. In litmus milk the dye is slowly reduced, and a whitish sediment but not curd formed; the final pH reaction is slightly alkaline. Positive results are observed in the following tests; nitrate reduction to nitrite, lipolytic activity, egg-yolk reaction, 2-ketogluconate production, citrate utilisation, aesculin hydrolysis and malonate test; and negative results in tests for indole formation, H2S production, oxidase, arginine dihydrolase activity, accumulation of poly-/3-hydroxybutyrate and hydrolysis of gelatin and cellulose.Growth will occur in a mineral medium with a carbohydrate carbon source and with (NH4)2S04 or KNO3 as sole nitrogen source. Vitamins and growth factors are not required. A wide spectrum of antibiotic activity is demonstrated.

P. antimicrobica has fungistatic and bacteriostatic properties. Therefore, it is clear the P.

antimicrobica contains one or more chemical compounds each having, singly or in admixture with at least one other of the compounds, fungistatic or bacteriostatic activity.

The fungicidal activity referred to above included antagonistic activity towards ordinary fungal cells and fungal mycelium.

The active compounds may be simply separated from P. antimicrobica by allowing the bacterium to remain in contact with an aqueous gel, the active substances diffusing from the bacterium into the gel.

Although I am not certain of the chemical nature of the active compounds in P. antimicrobica. I believe that they are novel compounds but they have not yet been chemically analysed and thus their chemical formulae and structures are not yet known.

In a first aspect the present invention provides a chemical compound separated from P.

antimicrobica and having fungistatic or bacteriostatic activity. The compound may conveniently be present in a composition comprising, in addition to the compound, an inert diluent or carrier.

In a second aspect the present invention provides a mixture of chemical compounds separated from the bacterium P. antimicrobica and having fungistatic or bacteriostatic activity.

The mixture may conveniently be present in a composition comprising, in addition to the mixture, an inert diluent or carrier.

The inert diluent or carrier comprised in compositions according to the invention may be agar.

It is envisaged that P. antimicrobica and the above described compounds will be useful in treating various substrates to prevent or control growth of fungi or bacteria ori or in the substrates.

In particular it is envisaged that the P. antimicrobica and above-described compounds will be useful, in human and veterinary medicine, for treating humans and animals to combat fungal and bacterial infections. E.g. it is envisaged that P. antimicrobica and the compounds will be useful in treating infections and ailments in animals and humans caused by fungal or bacterial infection, such as anthrax, thrush, breast abscesses, bovine abortion, tinea, horse ringworm and other animal ringworms.

Also, in particular, it is envisaged that P. antimicrobica and the above described compounds will be useful in treating plants to prevent or cure bacterial and fungal infections. E.g. it is envisaged that P.

antimicrobica and the compounds will be useful in preventing P. palmivora infection in cocoa fruit and similar species of plant.

Thus, in a third aspect of the present invention, the invention provides a method of suppressing or controlling fungal or bacterial growth in or on a substrate, comprising applying to the substrate a culture of P. antimicrobica or a compound or a mixture of compounds separated from P. antimicrobica and having fungicidal or bactericidal activity.

The substrate may be one in or on which fungi or bacteria are already growing or one in or on which fungi or bacteria would be liable to grow.

The following examples are given to illustrate the antagonistic activity of compounds contained in P. antimicrobica.

Example 1Fungicidal Activity Cultures derived from single spores of fungi were made in petri dishes containing potato dextrose agar, Czapak Dox agar or nutrient agar and allowed to grow to a diameter of about 25 mm at 25"C.

Using a sterile standard cork borer (No. 3), small cylinders of the agar were removed 20 mm from the edge of the fungus colony, leaving holes into which measured quantities of prepared test inhibitors were placed. In other tests, a loopful of 2-day old cultures of bacterium was applied to the agar (not in holes), again 20 mm from the fungus; or was applied direct to the mycelium near the edge of the fungal colony to study invasion. Plates were incubated and increase in radial growth of the fungus towards each hole or bacterial colony measured daily, noting any stoppage of fungal growth.

Pseudomonas aeruginosa, P. maltophilia, P. an tim ycetica and P. mors-prunorum were used as controls, P. an tim ycetica examined against bacteria only.

P. antimicrobica stopped the growth of all thirty-nine species of fungi which I examined. These species included members of the Genera Phytophthora, pythium, Penicillium, Botryodiplodia, Cylindrocarpon, Gueguenia, Ph om opsis, Fusarium, Asp ergillus, Candid, Cryptococcus, Gliocladium, Irichophyton, Microsporum, Arthroderma, Nannizzia, Merulius, Mucor (both mating strains of M.

hiemalls), Schizosaccharomyces, Trichothecium. Some species were more susceptible to this antifungal activity than others, their growth being stopped earlier (i.e. within 24 hours). P. aeruginosa suppressed the growth of 14 species and the other bacteria did not show inhibitor activity.

The bacterium was active in all the common laboratory agar media used. Cylinders of bacteriafree agar removed from positions 20 mm from the edges of 5 day-old colonies (3 mm in diameter) inhibited fungal growth. To obtain an active diffusate, the bacterium was cultured on a quarter of the surface area of plated agar (potato dextrose, nutrient or Czapak Dox) for 7 days.

The bacteria-free portion of the agar was then cut and put into sterile bottles each containing 10 ml of sterile distilled water and left for 24 hours at room temperature. The diffusate thus obtained was tested against Phytophthora palmivora and p. infestans and found active over a pH range of 3.4-8 (buffered) which were the values examined. Zoospores and mycelia of these fungi did not grow when they were used for inoculating agar containing the active diffusate.

Example 2-Mycolytic Activity When P. antimicrobica was inoculated in contact with fungal mycelium the filaments were often destroyed and evidently used as substrate. Often the invaded area became moist. When 3-day-old slide cultures were examined under the microscope, bacterial cells were seen inside the mycelial filaments just beyond the visible edge of the invading bacterial colony. Such mycelium did not grow when used for inoculation.

In some fungi, like Pythium graminicola, inhibition was followed by a collapse of mycelium while in others luxuriant aerial mycelium formed at the inhibited edge. The collapsed mycelium, which subsequently failed to grow when sub-cultured, appeared to indicate the point reached by a lethal concentration of the active substance secreted by the bacterium. When a plate colony of Botryodiplodia theobromae (5 cm in diameter) was ringed with a loopful of the bacterium, allowing a 5 mm-wide microbe-free zone between the two organisms, the edges of the black fungal colony began to turn transluscent within 3 days. Mycelial filaments were no longer evident in microscopical examination of the translucent areas, the surface of which was smooth and shiny; the condition suggested that the fungus was undergoing lysis due to the action of substances produced by the bacterium.

Example 3-Antibacterium Activity Three-day-old nutrient agar cultures of eight species of bacteria, each representing a different genus, and including both Gram-positive and Gram-negative organisms were separately suspended in 0.4 ml portions of sterile distilled water. Each of the suspensions was mixed with 40 ml of melted nutrient agar at 44-450C and plated in two petri dishes, one of which was reinoculated 2025 minutes later from a suspension of P. antimicrobica and from another of P. antimycetica at separate sites; the plate which was not reinoculated served as additional control. All plates were incubated at 250C and observed for possible zones of inhibition.

Within three days, clear zones had appeared which showed P. antimicrobica to inhibit the growth of P. morsprunorum, Corynebacterium michiganse (E. F. Smith) Jensen, Xanthomonas phaseoli (E. F.

Smith) Dawson, Bacillus cereus var. mycoides (Flugge), Strep tom yces griseolus (Krainsky) Waks. 8 Henr. and Sarcina flava de Bary; but Chromobacterium violaceum (Schroeter) Bergonzini and Protaminobacter ruber den Dooren de Jong. were not inhibited. The present species spread rapidly and produced yellowish-orange coloured diffusate in nutrient agar mixed with growing cells of P. morsprunorum, Corynebacterium michiganse or Proteminobacter ruber, and lesser amounts in the one with Streptomyces griseolus.

Example 4 Biological Control of a Fungal Disease Using P. antimicrobica (a) Inoculation techniques The effect of P. antimicrobica on the development of the black pod disease of cocoa caused by Phytophthora palmivora was studied in the laboratory and in the field. To provide a satisfactory method of assay, a technique was developed in which nearly every cocoa pod inoculated with this fungus became infected. A small cylindrical piece of the outer part of the pod husk, 4 mm in diameter, was removed to a depth of about 6 mm and replaced with similar tissue of equal dimensions from a pod infected with P. palmivora. A second technique involved a modification of that of Hislop (1962): Detached pods were chilled in an air conditioned room overnight, and transferred to a warmer room before spraying with zoospores.The necessary humid condition was provided by incubating the inoculated pod (suspended in a wire cage) inside a plastic bag into which was placed sterile wet cotton wool. The first technique ensured infection in all inoculated pods while the second was more closely related to conditions prevailing in the field.

(b) Inhibition of Infection in Detached Pods An inoculation hole was made in each of sixty detached pods from twelve Amelonado cocoa trees. The wounds thus created in twenty of these pods which were selected at random, were each sprayed, using a sterile atomiser, with about 0.1 ml sterile semi-liquid medium (pH 5.6) comprising 0.2 g potato dextrose agar in 30 ml distilled water. Twenty more pods were sprayed in a similar manner with a 24-hour-old culture of the bacterium in the same medium. Thirty to 40 min later all pods were plug-inoculated as described above and were kept at about 270C under polyethylene sheet to conserve moisture.

Another eight pods each with an inoculation hole at the proximal, lateral and distal parts were similarly sprayed, four with medium and four with the culture. These were plug-inoculated 48 hours later.

None of the pods sprayed with the culture of the bacterium became infected irrespective of whether they were inoculated soon after spraying or 48 hour; and they remained uninfected until they ripened and were discarded. However, all the pods with no bacterial treatment developed Phytophthora infection within 3 days at every inoculation site irrespective of position. The bacteriatreated pods showed no evidence that they were affected by the bacterium.

(c) Effect of Media on Inhibiting Activity in vivo One hundred Amelonado cocoa pods were used in a test in which one of two inoculation holes in each of twenty-five pods was filled with uninoculated nutrient broth (Oxoid) and the other hole with a culture of the bacterium in the same medium. The same treatments were given to the next twenty-five pods but using a semi-liquid medium made up of 0.2 g potato dextrose agar in 30 ml nutrient broth, instead of nutrient broth. The second lot of fifty pods were set up similarly using another pseudomonad, 46.B, isolated from the same source as the present one. Each of the one hundred pods was plug-inoculated immediately after applying the medium or the culture.

Disease development in the pods was again inhibited by treatment with the bacterium and this showed no appreciable difference between the two media used (Table 1). The bacterium, 46.B, caused some reduction of the disease and was more effective in the semi-liquid medium than in the liquid medium.

Table 1 Comparison of the development of Phytophthora palmivora in pods in the presence and absence of two bacteria.

Percentage of infection Liquid Semi-liquid Treatment medium medium Culture of present bacterium 0 8 Medium only 88 96 46.B culture 64 28 Medium only 96 84 Example 5-Reduction of Phytophthora Lesions in Unpunctured Detached Pods Using the semi-liquid medium described in Example 4, thirty detached cocoa pods were sprayed as follows:- 1. 10 pods: Uninoculated medium l followed by zoospore 2. 10 pods: Culture of the bacterium i suspension of P.

3. 10 pods: Uninoculated medium only. palmivora The pods were kept at 2 7--28 OC in polyethylene bags to conserve moisture and the number of lesions produced on them counted on the 4th day.

The bacterium again protected pods from Phytophthora infection (Table 2). Infection observed on two of the pods sprayed with the bacterium arose from the stalk end (i.e. by growth of mycelium from parent tree through the stalk).

Table 2 Reduction of zoospore infection by P. antimicrobica in uninjured pods P. antimicrobica Medium & culture 8 Medium only: zoospores zoospores nozoospores Number of diseased pods out of 10 7 2 2 Total number of lesions 28 2 2 The bacterium was not found on unsprayed pods freshly collected from the field; but it was still present on pods onto which it had been sprayed 1 6 days previously, although the concentration had declined considerably by this time (Table 3).

Table 3 Decline of cell-population pods after treatment with the bacterium Time after application Number of bacterial of bacterial culture cells per pod 6 hours 2000x106 4 days 100x 106 8 days 194X106 12 days 40x 106 16 days 32x 106 In similar tests carried out on undetached pods in the field, decline in bacterial population was faster than in the laboratory; but this could be due to rainfall which occurred during the period.

Example 6-Control of Black Pod Disease in the Field Towards the end of the main cocoa harvesting season when ripe cocoa pods were being picked, two groups of Amelonado cocoa trees bearing a large number of both diseased and healthy mature pods were selected on a farm which had a high incidence of black pod. The first group comprised twenty trees and the second nineteen. These carried ninety-one diseased plus one hundred and twelve healthy pods, and eighty-two diseased plus one hundred and twenty-eight healthy pods respectively.

Using a low pressure sprayer all uninfected pods on alternate trees in each group were individually sprayed, to run-off, with a culture of the bacterium prepared as described above in Example 4 in a semi-liquid medium. Pods on the remaining trees were left unsprayed as controls. After allowing 12 days for any latent infection to develop the sprayed pods were observed at 2-day intervals for new black pod infections until they were ripe and harvested.

The bacterial culture reduced the percentage of pods which became diseased and prevented further infection after the 1 6th day from the date of spraying with the bacterium; unsprayed pods in both replicates, however, continued to develop infection.

Claims (8)

Claims

1. A chemical compound separated from the bacterium P. antimicrobica, as described herein, and having fungistatic or bacteriostatic activity.

2. A mixture of at least two chemical compounds separated from the bacterium P. antimicrobica, as described herein, and having fungistatic or bacteriostatic activity.

3. A composition comprising a chemical compound according to Claim 1, or a mixture according to Claim 2, together with an inert carrier or diluent.

4. A composition according to Claim 3, wherein the inert diluent or carrier is agar.

5. A method of treating a substrate to prevent or inhibit fungal or bacterial growth or growth of fungal mycelium in or on the substrate, comprising applying to the substrate the bacterium P.

antimicrobica, a compound according to Claim 1 or a mixture according to claim 2.

6. A method according to claim 5, wherein the compound or mixture is applied in a composition comprising, together with the compound or mixture, an inert diluent or carrier.

7. A method according to claim 5 or 6, wherein the substrate is a material not forming part of a living animal.

8. A method according to claim 5 or 6, wherein the substrate is a living plant.