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WO1994003065A1 - LUTTE BIOLOGIQUE CONTRE LES INFECTIONS FONGIQUES A L'AIDE D'UNE SOUCHE DE $i(PSEUDOMONAS) - Google Patents

LUTTE BIOLOGIQUE CONTRE LES INFECTIONS FONGIQUES A L'AIDE D'UNE SOUCHE DE $i(PSEUDOMONAS) Download PDF

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Publication number
WO1994003065A1
WO1994003065A1 PCT/AU1993/000390 AU9300390W WO9403065A1 WO 1994003065 A1 WO1994003065 A1 WO 1994003065A1 AU 9300390 W AU9300390 W AU 9300390W WO 9403065 A1 WO9403065 A1 WO 9403065A1
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Prior art keywords
plant
fruit
digitatum
oranges
strain
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PCT/AU1993/000390
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English (en)
Inventor
Yong Huang
Brian James Deverall
Stephen Clive Morris
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The University Of Sydney
Commonwealth Scientific And Industrial Research Organisation
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Application filed by The University Of Sydney, Commonwealth Scientific And Industrial Research Organisation filed Critical The University Of Sydney
Priority to AU45521/93A priority Critical patent/AU4552193A/en
Publication of WO1994003065A1 publication Critical patent/WO1994003065A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Definitions

  • the present invention relates to the control of fungal infections in plants and plant products.
  • PAL phenylalanine ammonia-lyase
  • 2,4-D 2,4-dichlorophenoxyacetic acid.
  • the present invention relates to the use of an isolate of _ cepacia (ID 2131, described herein) which is capable of providing significant control of Pencillium di ⁇ itatum under both laboratory and packing line conditions .
  • the present invention also relates to a novel bacterial isolate (ID 2859, described herein) related to Pseudomonas ⁇ lathei capable of providing this activity.
  • These isolates should also be of use in protection against other fungal plant pathogens such as Fusarium crookwellense. Colletotrichium linde uthianum, P. italicum. and fungal agents which are tropical plant pathogens.
  • Isolates ID 2859 and ID 2131 were deposited with the Australian Government Analytical Laboratories of 1 Suakin Street, Pymble 2073, New South Wales, Australia in accordance with the provisions of the Budapest Treaty on 25 July 1991 under accession numbers N91/41766 and N91/41765 respectively.
  • ID 2859 does not appear to produce antibiotics and does not appear to rely on antibiotic production for its protective effect against fungal attack.
  • a novel Pseudomonas strain herein identified as ID 2859 there is provided a novel Pseudomonas strain herein identified as ID 2859.
  • a method for protecting a plant or plant product from fungal infection comprises exposing the plant or plant product to ID 2131 or ID 2859.
  • the plant or plant product is citrus fruit.
  • citrus fruit is post-harvest oranges.
  • the fungal infection is infection by _ digitatum.
  • the ID 2131 or ID 2859 is applied to the plant or plant product at a concentration of at least 10 5 colony forming units/ml (cfu/ml) .
  • the concentration is 10 8 cfu/ml to 10 9 cfu/ml.
  • the method comprises: washing the oranges with water; spraying the fruit with bacterial cells from a bacterial culture; and allowing the fruit to dry. The method is typically followed by sorting the fruit and then transporting or storing the fruit. Prior to sorting oranges are usually waxed, however,the wax "Britseal" (Milestone Chemicals Pty Ltd, Melbourne, Australia) has been shown to reduce the effectiveness of the bacterial treatment and therefore may be replaced with other waxing materials.
  • Alternative waxes which may be used include Shellac, Carnauba polyethylene and resin based waxes, (all available from Castle Chemicals Pty Ltd Australia) .
  • a combination of bacteria and a growth regulator such as 2,4-D preferably at a concentration of 250 ppm may be applied to the oranges.
  • the growth regulator is one which principally has auxin activity.
  • the fruit instead of spraying the fruit on the packing line, the fruit may be dipped in the bacterial cells from a bacterial culture either in the presence or absence of growth regulator, prior to loading onto the packing line.
  • derivatives of the antibiotic can be prepared using standard chemical techniques.
  • a method for protecting a plant or plant product from fungal infection comprises exposing the plant or plant product to the antibiotic of the third aspect.
  • an antifungal composition comprising an effective amount of strain ID 2131 or ID 2859 together with an agriculturally acceptable carrier or diluent compatible with maintaining viability of the strain.
  • composition may additionally comprise an effective amount of a growth regulator, which is typically a regulator having principally auxin activity.
  • an antifungal composition comprising an effective amount of the antibiotic substance of the third aspect or a derivative of it together with an agriculturally acceptable carrier or diluent.
  • _ cepacia ID 2131 inhibits germination of P_j_ digitatum and shows strong ability to protect wounded postharvest oranges from ⁇ ____ digitatum infection.
  • Treatment with £ ⁇ cepacia cells on unwounded Washington Navel and Valencia oranges under packing line conditions also significantly reduced Pj_ digitatum decay. Brown pigmentation appeared around some wound sites when the cell concentration was higher than 10 7 cfu/ml and PAL activity around injured sites was suppressed together with an inhibition of lignin synthesis.
  • a variant of strain ID 2131 in which the genes regulating the expression of the deleterious compound responsible for suppressing wound healing have been deleted. This variant could also be used in the methods of the invention.
  • FIG. 1 shows the effect of Pseudomonas cepacia concentration and time interval on control of Penicillium digitatum infection of Valencia oranges.
  • a A P___ cepacia applied 2 hr before P ⁇ . digitatum;
  • Fig. 2 shows the effect of Pseudomonas cepacia on PAL activity of wounded flavedo at healing sites of Valencia oranges at 25 or 30°C.
  • Water or P_j_ cepacia (1.3 x 10 9 cfu/ml) was applied to wounds made with sandpaper. Fruit were incubated at 96% relative humidity.
  • Fig. 3 shows a scanning electron micrograph of P_ ⁇ cepacia ID 2131 cells on a wound site.
  • Fig. 4 shows that where cells of P ⁇ cepacia ID 2131 are abundant on a wound site, spores of P_ s _ digitatum do not germinate.
  • Fig 5 shows that where cells of ______ cepacia ID 2131 are sparse on a wound site 9___ digitatum spores germinate.
  • Fig. 6 shows a trace of the purification of the effective antibiotic fraction from _____ cepacia ID 2131 cells collected from the reversed phase column on HPLC equipped with normal phase column.
  • Fig. 7 shows a 400 MHz X H NMR spectrum of the C 18 column bleed which was run in solvent CD 2 Cl 2 for the antibiotic from ⁇ ___ cepacia ID 2131 cells.
  • Fig. 8 shows a 400 MHz 1 H NMR spectrum of the active sample which was run in solvent CD 2 Cl 2 for the antibiotic from P_j_ cepacia ID 2131 cells.
  • Fig. 9 shows the off-resonance decoupled 13 C spectrum of the antibiotic compound. The solvent was CD 2 Cl 2 and the spectrum produced at 100 MHz.
  • Fig. 10 shows the main fragments of the antibiotic that may form during electronic impact mass spectrometry which are supported by high resolution mass spectrometry.
  • Fig. 11 shows the effect of the purified antibiotic produced by strain ID 2131 on PAL activity at healing sites of wounded flavedo Valencia orange at 30°C. Water or the antibiotic (50 ⁇ g/ml) was applied to wounds made with sandpaper. Fruit were incubated at 30°C with 96% relative humidity. Vertical bars represent the standard errors about the means of two replicates.
  • Fig. 12 shows the general structure of some terpenoid antibiotics isolated from microorganisms or plants.
  • Fig. 13a shows stereoscanning electron micrographs showing sparse cells of the strain ID 2859 of Pseudomonas glathei (upper) and germinated spores of Penicillium digitatum (lower) at wound sites on Washington navel oranges having been incubated at 25°C for 48 hr.
  • Fig. 13b shows stereoscanning electron micrographs showing the abundant bacterial cells (upper) and ungerminated fungal spores (lower) at wounded sites on fruit having been incubated at 30°C for 24 hr and then for a further 24 hr at 25°C.
  • Fig. 14 shows the effect of temperature on inhibition of Penicillium digitatum infection by strain ID 2859 of Pseudomonas glathei on wounded Washington navel oranges.
  • the fruit were wounded to 3 mm deep and inoculated with P. digitatum (6.7 x 10 5' spores/ml) , and then 2 hr later treated with the bacterium ID 2859 (1.7 x 10 9 cfu/ml) .
  • the fruit were either directly incubated at 25°C for 5 days or held at 30°C for 24 hr before being transferred to 25°C and incubated for a further 5 days before the symptoms were recorded.
  • isolates described herein can be cultured under the conditions described in the following Examples, and can be applied to plants or plant products using techniques such as those described in the following
  • compositions of the invention may be prepared using standard techniques and carriers and diluents as used in preparation of agricultural compositions.
  • the composition includes a growth regulator standard regulators may be used. Examples include 2,4-D and gibberellic acid.
  • the compositions comprise the bacterial isolates, the carriers and diluents are selected from those compatible with maintaining the viability of the bacteria.
  • Example 1 Inoculum.
  • a bacterial strain was isolated from the surface of Washington Navel oranges (Citrus sinensis Osback) at the Somersby research station, NSW Agriculture and Fisheries. After streak-separation and subculture on malt-extract agar (MEA) , the organism was identified from its fatty acid profile on a gas chromatography Microbial Identification System (Aerobe Library version 3.0, Microbial ID Inc, Newark DE) at NSW Agriculture and Fisheries Biological and Chemical Research Institute and produced the following fatty acid profile:
  • the bacterial cells were incubated routinely on MEA at 25°C for 5 days.
  • the cells were collected with a sterilized loop and suspended in distilled water. The concentration was determined as colony forming units per ml.
  • P. digitatum spores were collected from decayed Washington Navel oranges and cultured routinely on MEA at 25°C.
  • the spores for experiments were produced by wound- inoculating orange fruit, incubating the fruit at 25°C for 6 days and harvesting the conidia by brushing them into sterile distilled water containing 0.05% wetting agent 'Lisspol' .
  • the suspension was filtered through 4 layers of muslin and adjusted to the required concentration.
  • In vitro tests on the fungus. Tests on ___ ⁇ digitatum spore germination on slides were carried out by the same method as described in reference 7. For tests on agar plates 1 ml of Pj .
  • cepacia cell suspension (1.8 x 10 10 cfu/ml) was placed on the centre of MEA in Petri dishes and incubated at 25°C for 24 hr. The plates were then dusted with __P___ digitatum spores and incubated at 25°C for 48 hr before measuring diameters of inhibition zones around the bacterium. Then, pieces of agar in the inhibition zones and 0.5 cm away from bacterial colonies were removed and transferred onto fresh medium (MEA) . After being left at 5°C overnight the plates were dusted with _P___ digitatum spores. After incubation for another 48 hr at 25°C, diameters of inhibition zones around the agar pieces were measured.
  • fruit and their inoculation were harvested from Somersby station, NSW Agriculture and Fisheries. After being washed and sorted into uniform size, the fruit were sterilized with 80% ethanol and injured to a depth of 3 mm at four equidistant points around the equator. Generally, fruit were treated by pipetting 20 ⁇ l of a suspension of P. cepacia cells onto each injured site and 2 hr later, by pipetting 20 ⁇ l _____ digitatum spores. Alternatively as in Table 1-2, the bacterial cells were applied as sprays or dips to the whole fruit. As shown in Fig 1, fruit were treated by pipetting 20 ⁇ l _____ cepacia at different concentrations, and suspensions of P ⁇ . digitatum spores were dropped into wounded sites at the time intervals of 24 hr earlier, 2 hr earlier, coincident or 2 hr later relative to P_ ⁇ cepacia or water treatment .
  • P. cepacia cells were collected from MEA and transferred into nutrient broth. After incubation at 25°C for 72 hr the cultures were centrifuged at 10,000 g for approximately 15 minutes and the bacterial cells were resuspended in distilled water for spraying onto fruit.
  • a water-based wax 'Britseal' (Milestone Chemicals Pty Ltd, Melbourne, Australia) was used on some batches of both Washington Navel oranges and Valencia oranges throughout the experiments, being applied immediately after spraying fungicide or the bacterial suspension.
  • the experiments were arranged in a completely randomized design. Each treatment was replicated three times with sixty fruit for Washington Navel oranges and fifty for Valencia oranges per replication. Amounts of infection were measured by assessing presence or absence of green mold symptoms on individual fruit. Results were expressed and analysed as described above.
  • PAL assay was carried out in 10 ml sodium borate buffer (0.1M, pH 8.8) . Concentration of cinnamic acid was measured at 269 nm on a Cary 2200 spectrophotometer. PAL activity was expressed as ⁇ kat/mg protein. One katal is defined as the amount of enzyme converting 1 mole of substrate per second. Protein was determined by the method described by Bradford (2) . The experiment was repeated once. Observation under scanning electron microscope.
  • P_ s _ cepacia The protective ability of P_ s _ cepacia was influenced by its cell concentration and time interval between P. cepacia treatment and _?___ digitatum inoculation (Fig. 1) .
  • P. cepacia When applied 24 hr after inoculation with _____ digitatum, P. cepacia was ineffective.
  • E ⁇ _ cepacia When applied 2 hr after or at the same time, E ⁇ _ cepacia gave significant protection at high concentrations.
  • Application 2 hr before inoculation increased the effectiveness of protection at each concentration of the bacterium. Generally, increasing the bacterial concentration above an order of 10 7 cfu/ml substantially decreased P ⁇ _ digitatum decay.
  • Figure 3 shows a scanning electron micrograph of . cepacia cells on a wound site. Where cells of _____ cepacia were abundant, spores of P . ⁇ . digitatum had not germinated
  • a plant growth regulator 2,4-D commonly used on citrus fruit for long term storage and for keeping the button alive, did not inhibit ]______ digitatum infection and when mixed with P_j_ cepacia cells it did not significantly affect biocontrol on artificially wound inoculated fruit (Table 1-4) .
  • Tables 1-6 and 1-7 give results of trials under packing line conditions using carefully handled and unwounded fruit, showing significant biocontrol of P ⁇ _ digitatum in unwaxed fruit .
  • the effectiveness of biocontrol evident on unwaxed fruit seemed to be prevented by waxing.
  • Adding 2,4-D into the P ⁇ . cepacia suspension appeared to overcome the deleterious effect of waxing and was also beneficial in unwaxed fruit .
  • the isolate of P____ cepacia ID 2131 was strongly effective against P ⁇ . digitatum infection on postharvest oranges under both laboratory and packing line conditions.
  • the yield of these substances might be related to the number of bacterial cells. Increasing the number of bacterial cells significantly decreased green mold decay.
  • the protective strain of P_;_ cepacia produces both antibiotics and wound-healing inhibitive substances. It is therefore able to protect wounded fruit from E ⁇ _ digitatum infection through the antibiotics.
  • the promotive strain (7) of _ s _ cepacia may lack ability to produce antibiotics but may produce the substances which are responsible for suppression of PAL activity and lignin formation, thus favouring P ⁇ digitatum infection on undefended * wounded oranges.
  • the antifungal substances produced by the protective strain are different from the substances that suppress wound- healing.
  • Table 1-1 Effect of Pseudomonas cepacia (ID 2131) on germination of Penicillium digitatum spores.
  • P____ digitatum spore suspension 1.3 x 10 5 spores/ml
  • £ ⁇ _ cepacia cell suspension 1.9 x 10 8 cfu/ml
  • Germination was assessed on 100 spores in each of six replicate drops (20 ⁇ l) per treatment and germ tube length was measured from germinated spores among them.
  • Table 1-2 Effect of Pseudomonas cepacia (ID 2131) on inhibition of Penicillium digitatum infection on Washington Navel oranges using different application methods under laboratory conditions.
  • the injured fruit were dipped in the cell suspension for a few seconds.
  • a £_;_ digitatum spore suspension (2.6 x 10 5 spores/ml) was pipetted into wound sites (20 ⁇ l/wound) .
  • the fruit were held at 25°C in plastic bags for 5 days.
  • Table 1-3 Effect of cell concentration of Pseudomonas cepacia (ID 2131) on Penicillium digitatum infection and on responses at inoculation sites in injured Valencia oranges.
  • Table 1-7 Comparison of effect of Pseudomonas cepacia (ID 2131) and a fungicide on infection by Penicillium digitatum in unwounded Valencia oranges under packing line conditions 1 .
  • a few antibiotics produced by P. cepacia have been characterised (5) and one of them was found to be effective in control of Botrvtis cinerea and Penicillium expansum on apple (15) .
  • Electron impact (El) mass spectra were determined at 70 eV on a spectrometer which was equivalent to a Krato ⁇ MS 50 with a VG 3D8 control console at a source temperature of 250°C. A high resolution mass spectrum was obtained on the same instrument. The El mass spectrum was further confirmed by a comparison with a chemical ionisation (Cl) spectrum obtained on a Hewlett Packard 5890 series II Gas Chromatograph with a Hewlett Packard 5989A mass spectrometer as detector. The gas chromatograph was equipped with an HP1 column (crosslinked methyl silicone gum, 12 m x 0.2 mm x 0.33 ⁇ m film thickness) . The initial temperature was set at 60°C and gradually increased to 200°C at a rate of 15°C per minute. The temperature of the injector was 200°C, while the detector was at 250°C.
  • HP1 column crosslinked methyl silicone gum
  • HPLC High performance liquid chromatography
  • a Waters 600E apparatus equipped with a Waters UV detector 490E.
  • An infrared spectrum (IR) was measured on a FTS 20/80 (BIO-RAD, Digilab Division) .
  • the strain of P ⁇ _ cepacia was incubated in broth consisting of malt extract 20 g, peptone 1 g, dextrose 20 g in 1 L water. Conical flasks (1 L ) were charged with 200 ml of the above mixture. After having been sterilised at 121°C for 15 min and cooled down, the flasks were inoculated with the bacterial cells collected from agar surface on MEA and incubated at 120 rpm on a Gallenkamp orbital shaker at 25°C for 3 days.
  • the fermentation broth (50L) was centrifuged at 10000 g for 10 min and the supernatant liquid was collected and partitioned three times with equal amounts of chloroform at room temperature. After vacuum drying at 40°C, the remaining syrup (4.75 g) was taken up successively with 50 ml benzene followed by 35 ml benzene-ethyl acetate mixture (80:20) .
  • the residues were dissolved in acetone and checked for biological activity jin vitro.
  • the effective fractions (3.7064 g) were dissolved in acetone and applied onto silica gel TLC plates (20 x 20 cm, gel layer 250 ⁇ m, with 254 nm fluorescent indicator on glass, SIGMA) which were developed by ascending chromatography in chloroform/petroleum spirit/methanol (60:30:10) .
  • the plates were inspected under a UV lamp.
  • the silica gel corresponding to each fluorescent or quenching band, and the gel between the UV-responding bands, were scraped into a centrifuge tube.
  • the silica gel was then crushed and extracted three times with acetone and the extract checked for biological activity n vitro.
  • the effective fraction (17.5 mg) was dissolved in the mobile phase solvent (65% acetonitrile and 35% water) for HPLC purification.
  • the HPLC was equipped with a reverse phase (C 18 ) column (6 ⁇ m, 25 x 100 mm, Nova-pak, Millipore water) . Isocratic elution was employed with 65% acetonitrile and 35% water at a flow rate of 6 ml/min.
  • the UV detector was set at 230 nm. The fractions were detected by UV absorption and the eluates between each UV absorbing peak were collected separately and tested for bioactivity. The effective fraction appeared at 8 mm after sample injection.
  • the effective fraction (2.2 mg) was further purified on a HPLC semi-preparative column (lO ⁇ m, 9.4 x 500 mm, ODS-3 M9/50, Whatman) with acetonitrile/water (60:40) at a flow rate of 4 ml/min. The fractions were collected and tested as above. The effective fraction was obtained at 12 min after sample injection.
  • fractions obtained throughout the experiment were dried by rotary evaporation (40°C+_2) and assayed for antifungal activity in an agar diffusion test. Fractions were dissolved in acetone and placed in a well (0.5 cm in diameter) made in the centre of Petri plates containing 15 ml MEA. After having been left for 5 hr, plates were seeded by dusting J ⁇ _ digitatum spores and incubated at 25°C for 24 hr and inhibition zones around the wells were recorded.
  • Pyrrolnitrin had an Rf of 0.70 and gave a maroon colour with DSA.
  • the active fraction extracted from ID 2131 culture had by comparison an Rf of 0.43 and gave a light yellow colour with DSA on a TLC plate developed in CHC1 3 /petroleum spirit/MeOH (60:30:10) .
  • the HR mass spectrum provided the following fragmentations at, 306.2229 (C 19 H 30 O 3 ) , 288.2071 (C 19 H 28 0 2 ) , 264.2067 (C 17 H 28 0 2 ), 246.1661 (C 16 H 22 0 2 ) , and 237.1780 (C 15 H 25 0 2 ) .
  • a norditerpenoid structure a likely scheme for El and HR fragmentations is given in Fig. 10.
  • the 1 H - -"-H correlation spectrum also indicated the presence of protons at ⁇ 1.5 ppm and ⁇ 1.34 ppm overlapped by the contaminants from the C 18 column (Table 2-4) . It was likely that the proton at ⁇ 0.9 in the compound was from the column bleed since it also appeared in the column eluate as seen in its -""H NMR spectrum. The proton at ⁇ 1.35 coupled to that at ⁇ 0.9 indicated that this proton might also be from the column bleed, as was the proton at ⁇ 1.5 which was coupled to that at ⁇ 1.35. However, the other proton at ⁇ 1.5 was also coupled to those at ⁇ 2.5 and 4.05, indicating its relationship with these protons in the active compound.
  • the proton at ⁇ 1.35 was coupled to that at ⁇ 2.0, being another proton in the antibiotic.
  • the protons at ⁇ 3.45 and 6.3 were coupled to that at ⁇ 0.9, indicating that they were also impurities as was the hydrogen at ⁇ 9.5 which was coupled to that at ⁇ 3.45 ppm.
  • Antibiosis is hypothesised to be a major mechanism of biocontrol according to the observation of inhibition zones on agar plates in many interactions between plant pathogens and biocontrol agents (32) .
  • the antifungal compound extracted from the fermentation broth of the strain ID 2131 of P. cepacia had a different structure to the previously described antibiotics from this species, pyrroles, pseudanes and cepacins.
  • the data resulting from the current experiments suggests that it is a cyclic nor-diterpene (C19) .
  • Cyclic terpenes as a class of natural products display a bewildering array of structural types. Over a hundred different carbon frameworks have been detected so far (31) .
  • gibberellins plant growth hormones, which were first isolated from a fungus Gibberella fujikuroi , the cause of
  • Pleuromutilin is an antibacterial substance which was isolated from a Basidiomycete , Pleurotus mutilus (33) . Fusidic acid, another antibiotic, was extracted from Fusidium coccineum
  • the antibiotic extracted from the growth culture of the strain ID 2131 was strongly inhibitive to infection by P. digi tatum on oranges. This was particularly distinctive when the pathogen had been applied to wounded sites 24 hr earlier, under which conditions the antagonistic cells were not able to provide their protection. This indicated that the antibiotic effect observed in vi tro could also be seen in the inhibition of P. digi tatum infection in vivo .
  • the purified antibiotic did not suppress host PAL activity, indicating that the inhibition of P. digi tatum spore germination and suppression of host wound healing by the bacterial cells of the strain ID 2131 and its growth culture came from different compounds, which must be controlled by different genes. Therefore, the genes regulating the production of the deleterious compound responsible for suppressing host wound healing should be capable of being deleted and the biocontrol effect by this strain thereby improved.
  • Such genetic manipulations have been done on Agrobacterium radiobacter K84 that possesses a transferable region on its plasmid that often results in decrease or loss of biocontrol effect (25) . Biocontrol effect has been significantly improved after a deletion of this region, with the production of a new strain K1026 (28) .
  • Table 2-1 Effect of heating the extracted compound at different temperatures on growth from Penicillium digitatum spores 1 .
  • Table 2-5 Effect of cells of Pseudomonas cepacia (ID 2131) and extract from their culture on decay in wounded oranges inoculated with Penicillium digitatum 1 .
  • the antibiotic 20 ⁇ l/wound (50 ⁇ g/ml) was applied to the wounded sites on Valencia oranges 10 hr after P. digitatum (5.9 x 10 5 spores/ml) inoculation. The fruit were incubated at 25°C for 5 days after P. digitatum inoculation.
  • Penicillium digitatum and V_____ italicum infection on citrus fruit (21) are examples of antagonistic bacteria.
  • their effect is- poor and less than that of antagonistic bacteria.
  • a bacterium was isolated from Valencia oranges and identified (ID 2859) as a strain related to Pseudomonas glathei by the MicroStationTM System, Biolog method (Anon, 1992 Release 3.01; Biolog Inc Hayward CA) . Isolate ID 2859 was also analysed for fatty acid profile on an HP 5898A Microbial Identification System at the NSW Agricultural and Fisheries, Biological and Chemical Research Institute.
  • the bacterial cells were collected from MEA and incubated in nutrient broth (nutrient broth 8 g, yeast extract 5 g, dextrose 10 g in 1 L water) on a shaker (120 rpm) at 25°C for 72 hr. The fermentation broth was centrifuged at 10000 g for 15 min and the bacterial cells resuspended in distilled water.
  • nutrient broth nutrient broth 8 g, yeast extract 5 g, dextrose 10 g in 1 L water
  • the fermentation broth was centrifuged at 10000 g for 15 min and the bacterial cells resuspended in distilled water.
  • P. digitatum spore suspension was prepared as in Example 1.
  • Valencia oranges were artificially injured (3 mm deep) and inoculated with a P ⁇ digitatum spore suspension (3.1 x 10 5 spore/ml) at a rate of 20 ⁇ l per wounded site. Two hr later, the fruit were placed in plastic bags and divided into four groups. One group was kept at 25°C for 4 days, the other three were incubated at 30°C for a period of 24 hr, 48 hr or 72 hr, respectively. The fruit were then kept at 25°C for 6 days after being taken out from the 30°C environment. Water treatment was used as a control by the same method.
  • Results ID 2859 did not produce any surrounding inhibition zone in the fungus at either 25 or 30°C conditions. (Table 3-1) .
  • the strain of _X_ glathei did not produce visible inhibition zones in the fungus on agar plates. It gave an inhibition of P ⁇ digitatum infection on oranges at 25°C if the fruit were inoculated with P ⁇ digitatum 2 hr later than its application, but not if inoculated 2 hr earlier. It strongly protected the wounded fruit from P. digitatum infection if fruit were healed at 30°C for a period after inoculation with P___ digitatum and treated with the bacterium.
  • Valencia oranges were inoculated with P ⁇ digitatum (3.1 x 10 5 spore/ml) and two hr later treated with the bacterial cells (3.9 x 10 10 cfu/ml) . After being left for another hr at 25°C, the fruit were incubated at 30°C for 24 hr, 48 hr or 72 hr, respectively. Fruit were then kept at 25°C for 6 days after being taken out from the 30°C environment.
  • the bacterium isolated from Valencia oranges and identified (ID 2859) as a strain related to Pseudomonas glathei by the method of MicroStationTM System, BioLog (Anon, 1992) was used.
  • Bacterium (ID 2131) was used for comparison in some experiments.
  • the bacterial suspensions were prepared by culture in nutrient both at 25°C for 48 hr.
  • the bacterial cells were collected by centrifugation at 10,000 g and suspended in distilled water.
  • the concentration of antagonists was determined as colony forming units per ml.
  • Penicillium digitatum was isolated from decayed Washington navel oranges and then cultured routinely on malt extract agar (MEA, malt extract 20g, dextrose 20g, peptone lg in IL distilled water) at 25°C. Inocula for experiments were produced by wound-inoculating orange fruit, incubating the fruit at 25°C for 6 days and then harvesting the conidia by collecting them on sterilised cotton swabs for dusting, or by brushing them into sterile distilled water containing 0.05% wetting agent Lissapol (ICI, Sydney, Australia) for spraying. The suspension of Pj. digitatum spores was filtered through four layers of muslin and adjusted to the required concentration.
  • MEA malt extract 20g, dextrose 20g, peptone lg in IL distilled water
  • the plates were immediately dusted with P ⁇ _ digitatum spores and incubated at 30°C for 72 hr before being moved to 25°C for a further 48 hr.
  • the growth of the fungus around the bacterial colonies was investigated under both temperature conditions.
  • Washington navel oranges were obtained and artificially injured to a depth of 3 mm using a sterile nail through a cork.
  • the fruit were inoculated with P. digitatum (6.7 x 10 5 spores/ml) and, 2 hr later, treated with the bacterial strain ID 2859 of ⁇ X_ glathei (1.7 x 10 9 cfu/ml) by pipetting 20 ⁇ l of the bacterial suspension into the wounded site. They were either directly incubated at 25°C or held at 30°C for 24 hr before being transferred to 25°C for 5 days.
  • the bacteria (ID 2859) grew normally on MEA at both 25°C and 30°C and did not inhibit surrounding fungal growth under these conditions. £_;_ digitatum did not grow on MEA at 30°C until the plates were removed to 25°C.
  • Figure 13a shows that bacterial cells of _?___ glathei (ID 2859) were sparse and fungal spores had germinated after inoculated fruit were directly incubated at 25°C for 48 hr. However, when the fruit were incubated at 30°C for 24 hr before being moved to 25°C the bacterial cells became abundant and the fungal spores did not germinte (Figure 13b) .
  • the bacterial strain ID 2859 was ineffective in biocontrol of fruit decay if it was applied 2 hr after the fungus ( Figure 14; Table 4-1) .
  • the biocontrol effect of ID 2859 significantly increased when intervals between applying the bacterium and then the fungus were increased from 2 to 24 hr (Table 4-1) .
  • the biocontrol effect brought about by ID 2859 was inferior to that by ID 2131 unless the bacteria were applied 10 hr before the f ngus.
  • Strain ID 2859 was very effective in biocontrol even when applied 4 hr after the fungus, where fruit had been held at 30°C for 24 hr before being returned to 25°C. It was totally effective where fruit had been held at 30°C for 48 hr. It was superior to strain ID 2131, under these conditions ( Figure 14; Table 4-2) . Both strains prevented infection when applied 2 hr before the fungus where fruit were held at 30°C for periods of 24 to 72 hr.
  • the bacterium ID 2859 could be readily recovered from all sites where it and the fungus had been applied (Table 4-3) .
  • _P___ digitatum could also be recovered from most sites where it had been applied alone or with the bacterium. _____ digitatum did not cause any symptoms, however, at wounded sites where the bacterium was present.
  • the sites inoculated with both organisms were sampled for isolates of fungi and bacteria, then washed with 70% ethanol and water and reinoculated with P_;_ digitatum, no bacterial cells were recovered and 77.5% of the sites developed green mould decay another 4 days later.
  • the bacterial isolate ID 2859 of P___ glathei gave a moderate biocontrol of P____ digitatum infection on postharvest oranges at normal conditions (25°C) . Its protective ability was significantly improved if the inoculated fruits were incubated at 30°C for 24 hr. It has already been established that wounded oranges can heal faster at 30°C and become less susceptible to P. digitatum infection, however, a satisfactory reduction of Penicillium decay requires at least 72 hr healing at 30°C and >96% RH (36) . This was confirmed in this experiment. Exposing the fruit to such a high temperature for 72 hr can result in a reduced storage life due to high respiration rate (37) .
  • the bacterium ID 2859 was readily recovered from the inoculated sites as was _P____ digitatum. This indicates that the bacterium did not eradicate _____ digitatum from the fruit but rather inhibited its infection of the fruit. Washing the wound sites inoculated with the bacterium before re-inoculating with the pathogen resulted in no later recovery of the bacterial cells and a significant loss of biocontrol at these sites. This suggests that the mode of action by this antagonist requires the presence of the bacterial cells and may involve a nutrient/space competition. Such a mode of action has also been suggested for biocontrol by yeast antagonists on apples (40) .
  • P. digitatum spores germinate well in aqueous extracts of orange rind or juice, or in sugars in the presence of phosphate buffer; but very poorly in water with or without the buffer or in sugars without the buffer. It has been concluded, therefore, that P. digitatum spores do not germinate in nature until they reach unhealed injured areas on the fruit.
  • exopolygalacturonase In order to infect the fruit, exopolygalacturonase (exo-PG) needs to be induced from P____ digitatum spores by the substrate pectic acid which is formed through the activity of endogenous pectinmethylesterase (PME) on pectin in the damaged fruit cells.
  • P_j digitatum infection by the bacterium ID 2859; 1) competing for nutrients or for substrates like pectic acid, or for oxygen with __ ⁇ digitatum spores; 2) physically separating P_j_ digitatum spores from the injured tissue on fruit; 3) deactivating enzymes with exo-PG or PME activity.
  • the non-antibiotic producing antagonist ID 2859 requires a much longer time than the antibiotic producing antagonist ID 2131 (at least 10 hr) to establish at the infection court before inoculation of the pathogen. This observation suggests that ID 2131 established biocontrol more quickly due to antibiotic production than does ID 2859 which does not produce an antibiotic.
  • the biocontrol by ID 2859 could be significantly improved if the inoculated were held at 30°C for a period. At 30°C for 24 hr, not only do the fungal spores not germinate but the bacterial cells have become much more numerous, indicating that they had multiplied quickly and established themselves during this time. As a consequence of establishment, the bacteria may be able to compete for nutrients with the pathogens and/or separate the pathogen spores from the injured tissues, from which the fungal spores presumably must obtain necessary nutrients for their germination. Table 4-1. Effect of the strains ID 2859 of Pseudomonas glathei and ID 2131 of Pseudomonas cepacia on control of Penicillium digitatum infection on wounded oranges at 25°C .
  • Table 4-3 Occurrence of fungal infection and recovery of fungal colonies and viable bacterial cells at sites inoculated with the strain ID 2859 of Pseudomonas glathei and with Penicillium digitatum, and in control
  • the present invention is of use in agriculture such as in the fruit industry for protection of fruit from fungal decay.

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Abstract

Des souches de Pseudomonas sont utilisées selon un procédé de lutte biologique contre les infections fongiques dans des plantes et des produits végétaux tels que les agrumes. Une nouvelle souche de Pseudomonas pouvant être utilisée selon ce procédé de lutte biologique est décrite, ainsi qu'une nouvelle substance antibiotique qui a été isolée d'une autre souche de Pseudomonas. La substance antibiotique présente une activité antifongique.
PCT/AU1993/000390 1992-07-31 1993-07-30 LUTTE BIOLOGIQUE CONTRE LES INFECTIONS FONGIQUES A L'AIDE D'UNE SOUCHE DE $i(PSEUDOMONAS) WO1994003065A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010018371A2 (fr) * 2008-08-13 2010-02-18 University College Cardiff Consusltants Ltd Agent antimicrobien et son procédé de production
WO2020187822A1 (fr) * 2019-03-18 2020-09-24 Futureco Bioscience, S.A. Souche de pseudomonas sp., composition la comprenant et ses utilisations
CN114397296A (zh) * 2021-12-29 2022-04-26 上海中医药大学 一种鉴定人参参龄的方法
CN115820431A (zh) * 2022-10-18 2023-03-21 天典(广东)生物科技有限公司 一株产核酸酶p1的橘青霉及其应用

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WO1990001327A1 (fr) * 1988-08-03 1990-02-22 Wisconsin Alumni Research Foundation INOCULANT BIOLOGIQUE EFFICACE CONTRE LES $i(APHANOMYCES)
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EP0255774A1 (fr) * 1986-07-28 1988-02-10 Stine Seed Farm, Inc. Produits et méthodes agricoles
WO1989007891A1 (fr) * 1988-02-24 1989-09-08 The United States Of America, As Represented By Th Regulation biologique de la pourriture grise dans les fruits a pepins a l'aide de acremonium breve
EP0350244A2 (fr) * 1988-07-01 1990-01-10 Stine Seed Farm, Inc. Procédé pour protéger les plantes contre les nématodes
WO1990001327A1 (fr) * 1988-08-03 1990-02-22 Wisconsin Alumni Research Foundation INOCULANT BIOLOGIQUE EFFICACE CONTRE LES $i(APHANOMYCES)
EP0444664A2 (fr) * 1990-02-28 1991-09-04 Suntory Limited Gènes résistants à l'acide fusarique
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010018371A2 (fr) * 2008-08-13 2010-02-18 University College Cardiff Consusltants Ltd Agent antimicrobien et son procédé de production
WO2010018371A3 (fr) * 2008-08-13 2010-04-08 University College Cardiff Consultants Ltd Agent antimicrobien et son procédé de production
GB2475440A (en) * 2008-08-13 2011-05-18 Univ Cardiff Antimicrobial agent and method for the production thereof
GB2475440B (en) * 2008-08-13 2013-05-29 Univ Cardiff Antimicrobial agent and method for the production thereof
WO2020187822A1 (fr) * 2019-03-18 2020-09-24 Futureco Bioscience, S.A. Souche de pseudomonas sp., composition la comprenant et ses utilisations
CN114397296A (zh) * 2021-12-29 2022-04-26 上海中医药大学 一种鉴定人参参龄的方法
CN115820431A (zh) * 2022-10-18 2023-03-21 天典(广东)生物科技有限公司 一株产核酸酶p1的橘青霉及其应用
CN115820431B (zh) * 2022-10-18 2023-08-01 天典(广东)生物科技有限公司 一株产核酸酶p1的橘青霉及其应用

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