Classifications

• • 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
  • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
  • A01N63/32—Yeast

Definitions

• biocontrol techniques in horticulture have not been commercially successful and one reason for this is that there is often a lack of reproduceability between trials in vitro and in the field. Also when compared to chemicals biological techniques suffer because of inoculum production, application and cost. Chemicals are also often preferred on the grounds of efficacy because chemicals generally work irrespective of environment or inoculum potential. Chemicals also are easier to apply to target plants and also a greater range of pathogens may be controlled.
• U.S. Patent 4,878,936 describes the use of B cereus ATCC 53522 as having biocontrol activity and which produces a fungicide active against Phytophera megasperm .
• Russian Patent SU 237480 reference is made to a strain of B cereus useful as a biocontrol agent for protecting plants against insects.
• compositions containing biosynthetic pesticidal products obtained from Bacillus bacteria selected for B thuringiensis, B sphaericus, B popilliae, B cereus, B lentimorbus or B ⁇ iboInstitutsis. These bacteria may also be provided with a low melting point polyester as described in Patent EP 145087.
• Patent Specification JP 59082085 there is described a method of controlling harmful insects by use of a biocontrol agent containing Bacillus subtilus, Bacillus coagulans, Micrococcus luteus, Bacillus stearothermophilus , Clostridium pasteurianum, Clostridium aminovalericum, Clostridum thermo- saccharolyticum and Thermoactinomyces vulgaris.
• Microbiology 52 552-555 (1986) which describes a variety of lactic acid bacteria isolated from plant surfaces and plant associated products which were found to be antagonistic to the phytopathogens Xanthomonas campestris, Envinia catotovora and Pseudomonas syringae.
• treatment of bean plants with a Lactobacillus planetarium strain before inoculation with Pseudomonas syringae caused a significant reduction of the incidence of disease.
• natural isolates which may be found on leaf surfaces of fruit and cultivate such natural isolates for their use as biocontrol agents when applied to crops or fruit and other plants.
• the natural isolates may be removed from the leaf surfaces and cultivated in suitable media.
• culture media may be dried and ground into a powder before being incorporated in a carrier.
• the culture media may be mixed with wax such as a water or paraffin/mineral oil base.
• the infected fruit may be dipped into a solution containing the natural isolate.
• the biocontrol agent of the invention may include or be derived from:
• the yeast component is suitably useful for producing a leavening or rising action in a sourdough which are doughs in which flour (for example, rye flour) is fermented by lactic acid bacteria rather than by bakers yeast.
• Sourdoughs are described, for example, by Oura et al. in Economic Microbiology, Volume 7 entitled "Fermented Foods" edited by A. H. Rose pages 123-134.
• Such yeasts may be selected from Candida krusei, Saccharomyces cerevisiae, Saccharomyces exiguss and Pichia saitoi.
• any yeast could be utilised which is effective in combination with an appropriate bacterial component and a suitable substrate to produce as main products lactic acid, acetic acid, ethanol and carbon dioxide which is the hallmark of sourdough fermentation.
• a glycolytic pathway is characterised by homofermentative lactic acid bacteria via aldolase and a heterofermentative lactic acid bacteria pathway for glucoses and pentoses via phosphoketolase.
• Saccharomyces exiguss is alternatively known as Torulopsis holmii.
• Other yeasts that may be used include yeasts of the genus Saccharomyces generally which include as mentioned above S. cerevisiae, S. exiguss, S. inusitatus and S. uvarum.
• the bacterial component of the symbiotic mixed culture may be selected from Lactobacilli as well as members of the genera Leuconostoc, Pediococcus and Streptococcus of the family Streptococcaceae. Different species of Lactobacilli may be used depending on th eir ability to use different substrates.
• Typical lactic acid bacteria may be selected from L. plantarum, L. casei, L.
• L. leichmannii L. brevis (especially var lindneri)
• L. fermentum L. pastorianus
• L. bucheri L. acidophilus
• L. farciminis L. alimentarius
• L. fructivorans L. viridescens
• L. cellobiosus L. solivarius.
• Other bacteria that may be used include Pediococcus cerevisiae, P. acidilactici as well as Citrobacter spp or Micrococcus spp.
• An especially preferred bacteria for use in the biocontrol agent of the invention are Lactobacillus sanfrancisco which are used in the San Francisco sourdough "French bread" process. These bacteria are indifferent to oxygen and do not use carbohydrates other than maltose as a carbon source.
• L. sanfrancisco form a symbiotic relationship with Saccharomyces exiguss which mainly performs the leavening function.
• L. casei ssp casei which was characterized by Rogose et al. in 1953.
• differentiation between L. buchneri and L. parabuchneri can be very difficult and it is within the scope of the invention to provide a mixed starter culture which includes L. buchneri in substitution of L. parabuchneri and other strains of L. casei for example in substitution of L. casei ssp casei. Differentiation may also be extremely difficult between L. parabuchneri, L. buchneri and L. brevis and L. brevis may also be substituted for L. parabuchneri or L. buchneri.
• a sample of the mixed culture was forwarded to Kluyver Laboratory of Technology Delft Technical University in The Netherlands and Lactobacilli strains (i), (ii) and (iii) were identified.
• the yeast component of the mixed culture was also determined as Saccharomyces cerevisiae Meyen ex Hansen by
• the substrate utilised in the mixed culture may comprise any preparation derived from cereals such as flour and in particular rye flour or white flour which may include unbleached white wheat flour. Molasses may also be utilised as a substrate.
• the substrate may also be mixed with water which is suitably "pure water” comprising bottled rainwater or sterilised Milli-Q filtered water.
• the pH of the mixed starter is suitably in the range 3.0-6.0 and more suitably 3.0-4.5.
• Fermentation may be initiated by mixing equal parts flour and water to form a paste, and this is then added at a suitable ratio (e.g. 1:1) to starter culture. Incubation may occur over 5-48 hours but more suitably 24 hours.
• a suitable ratio e.g. 1:1
• the biocontrol product of the invention is compatible with sustainable agriculture and free from health risks which is not the case with conventional chemical or antibiotic fungicides as discussed above which are disadvantageous because of: (a) high oncogenic risks;
• biocontrol agent of the invention may also include other components so as to facilitate its mode of action in a desired application.
• one component may include a carrier which may help to establish and maintain antagonistic populations of the biocontrol micro ⁇ organism on the target surface.
• This carrier may be a lipid such as wax or oils inclusive of coconut oil, vegetable oil, olive oil, canola oil or emulsions inclusive of fish oil emulsion. Suspensions or slurries may also be used consistent with the above criteria.
• One suitable carrier is an emulsified vegetable' oil identified by the trade mark CODACDDE.
• Another lipid carrier is identified by the trade mark NU-FTLM 17. The use of the lipid may also enhance dispersability and may also be used as an adhesive to the target surface.
• a thickener which is suitably a gum such as natural or modified gums inclusive of algin, carrageenan, xanthan and tragacanth gums.
• a gum may also be used as an emulsifier or stabiliser.
• pathogens of aerial surfaces of crop plants or crop products which may be subjected to the biocontrol agent of the invention include mainly fungal pathogens i.e. Colletotrichum (anthracrose) , Cercospora
• micro-organism antagonism enhancer(s) which may include MgO, MgCO 3 and/or CaCl 2
• pathogen inhibitors which may include tea polyphenols (especially epicatechin gallate and epigallocatechin gallate) as well as antioxidants and substances to facilitate production, storage and/or application of the agent.
• the sourdough biocontrol products of the invention may be formulated for commercial purposes in any suitable manner and thus may be freeze dried to improve storage life.
• the biocontrol agent of the invention may be applied to the target before, during and after harvest.
• the SDBS starter culture was stored at 4°C before use. To reactivate, equal parts of "Kialla” organically grown unbleached white flour and Milli-Q filtered water were mixed to form a smooth paste. The paste was then combined with the starter culture (1:1) sometimes it may be necessary to fully decant off the "clear layer" of accumulated acetic acid, lactic acid and ethanol from the top of the starter culture when this is more than V6 of the total volume. The culture was then incubated in a loosely covered glass jar at room temperature (23-30°C) for mostly 24 hours but up to five days.
• the SDBS mixture appears to act fungistatically to inhibit or retard the development of anthracnose on naturally infected common mango fruits.
• the fungistatic effect of the treatment can be eliminated when the mixture is removed from the fruit surface by washing.
• the effect can be enhanced by prolonged exposure to the actively growing (wet) mixture by repeated dipping or by the addition of the spreader - sticker formulations Nufilm 17 and Codacide.
• the SDBS starter culture originated from a sourdough starter which was developed by Mrs Edith Aylward of Roys Road, Palmwoods, Queensland, Australia.
• the starter initially comprised a combination of intact grapes of the Cabernet variety and a commercial starter obtained from Germany called "BACK FERMENT” which was utilised in rye bread manufacture and such commercial starter was mixed with water and unbleached organic white flour (“KIALLA”) in equal volumes.
• KIALLA unbleached organic white flour
• Lactobacillus casei ssp caesi grows well on Lactose LB medium, while the other isolates either do not grow or only grow very slowly and poorly on this medium.
• This medium to estimate the proportion of L. casei ssp casei in the total count.
• yeast population growth seems to be very sensitive to both oxygen availability and organic acid and ethanol concentration. If yeast numbers are low in the starter at the beginning of the fermentation (in our experience due to prolonged growth and/or storage under anaerobic conditions), the population will not grow and very little ethanol production will occur. When reasonable numbers are present > 10 3 per ml, the population seems to be active in the first 24 hours of fermentation producing ethanol. This can be seen in the fermenting mix as it becomes very frothy. As the fermentation proceeds, acid accumulation and possibly oxygen depletion will limit yeast growth and activity. When this occurs the mixture stops frothing and dies down again.
• LLA medium comprised Lactic Acid Bacteria Media using lactate. This medium comprised. 0.03% Tween 80, 1.5% fresh yeast extract, 0.6% tryptone and 2.0% lactose which was adjusted to pH 5.45 with agar.
• SLA medium is the same as LLA medium except sucrose is utlised instead of lactose at the same concentration.
• MLS medium is the same as LLA medium except maltose is used instead of lactose at the same concentration.
• RSYM medium comprised 10% raw sugar, 0.05% (NH ⁇ 2 HPO 4 1.5% fresh yeast extract and 2% agar. Water is added to 100% and the pH adjusted with IN HCl to pH 6. The medium should be autoclaved before use.
• SYM medium is the same as RSYM with sucrose being used instead of raw sugar.
• MEA is a conventional malt extract medium obtained from Oxoid Australia.
• PDA is a conventional potato dextrose medium also obtainable from Oxoid Australia.
• RAA is a conventional rogosa agar medium obtainable from Oxoid Australia.
• Molasses media compriess 1%, 2%, 5%, 10% or 20% molasses in water which is autoclaved before use.
• MRS medium is also obtainable from Oxoid Australia where 52 g of MRS broth is dissolved in 1000 ml of MilliQ H 2 O and 20 g of agar is added. The mixture is autoclaved at 121 °C for 15 minutes.
• Table 6 shows pH profiles of the standard fermenation at 30°C.
• Runs I and 2 yeast numbers were very low at the beginning of fermentation.
• Tables 10 and 11 illustrate experiments which were conducted using a protocol similar to that described in regard to Table 1 which measures in Table 10 the mean diameter of lessions in cm on mango fruit naturally infected with anthracrose over 17 days post treatment with the biocontrol agent of the invention which was prepared as set out in the protocol corresponding to Table 1 above.
• Table 11 illustrates the increase in density of lesions on naturally infected mango fruit over a 17 day period after treatment.
• Table 12 refers to reductions in anthracnose development in mangoes and avocado after post harvest treatment with SDBS.
• FIG 1 refers to the development of anthracnose on naturally infected mangoes after 7 days incubation at RT as discussed above in relation to Table
• FIG 2 refers graphically to the effect of sticker-spreaders on the development of anthracnose on naturally infected common mangoes
• FIG 3 is a flow diagram illustrating a proposed method of manufacture of the biocontrol agents of the invention on a commercial scale.
• FIG 4 is a graph illustrating in graphical form the data set forth in Tables 11-12.
• FIG. 5 is a graph illustrating in graphical form the data set forth in
• inventive concept refers to the realisation that sourdough starter formulations surprisingly have a biocontrol capability especially in relation to fungal diseases of plants.
• This is exemplified by application of the biocontrol agent of the invention to mangoes and avocado which were infected with Colletotrichum gloeosporioides and Collectotrichum acutatum.
• a process for production of a biocontrol agent for use in biocontrol of fungal diseases in fruit or plants including the steps of:
• FIGURE 1 The effects of SDBS post harvest dip on the development of anthracnose on naturally infected mangoes after 7 days incubation at room temperature.
• FIGURE 2 The effect of 3 dips and sticker-spreader on the development of anthracnose on naturally infected common mangoes.
• FIGURE 4 pH and organic acid profiles of the standard fermenation at 30 °C (Mean of 3 replicate fermentations, Run 2N).

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• Life Sciences & Earth Sciences (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• Mycology (AREA)
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• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Pest Control & Pesticides (AREA)
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• Agronomy & Crop Science (AREA)
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• Agricultural Chemicals And Associated Chemicals (AREA)
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• 1994
  • 1994-03-10 JP JP6519390A patent/JPH08507497A/ja active Pending
  • 1994-03-10 CA CA002157904A patent/CA2157904A1/en not_active Abandoned
  • 1994-03-10 WO PCT/AU1994/000110 patent/WO1994019950A1/en not_active Application Discontinuation
  • 1994-03-10 NZ NZ262853A patent/NZ262853A/en unknown
  • 1994-03-10 EP EP94910276A patent/EP0688169A4/de not_active Withdrawn

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