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WO2011115798A2 - Traitement antimicrobien pour des graines et des pousses - Google Patents

Traitement antimicrobien pour des graines et des pousses Download PDF

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Publication number
WO2011115798A2
WO2011115798A2 PCT/US2011/027719 US2011027719W WO2011115798A2 WO 2011115798 A2 WO2011115798 A2 WO 2011115798A2 US 2011027719 W US2011027719 W US 2011027719W WO 2011115798 A2 WO2011115798 A2 WO 2011115798A2
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Prior art keywords
seeds
seed
levulinic acid
sds
composition
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PCT/US2011/027719
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English (en)
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WO2011115798A3 (fr
Inventor
Ronald Walcott
Michael Doyle
Tong Zhao
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University Of Georgia Research Foundation, Inc.
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Priority to US13/510,724 priority Critical patent/US20130005811A1/en
Publication of WO2011115798A2 publication Critical patent/WO2011115798A2/fr
Publication of WO2011115798A3 publication Critical patent/WO2011115798A3/fr

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    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing within the same carbon skeleton a carboxylic group or a thio analogue, or a derivative thereof, and a carbon atom having only two bonds to hetero atoms with at the most one bond to halogen, e.g. keto-carboxylic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/08—Immunising seed

Definitions

  • 61/314,922 entitled “TREATMENT FOR ALFALFA SEEDS AND SPROUTS” filed on March 17, 2010, and to U.S. Provisional Patent Application Serial No.: 61 /316,582 entitled “EFFICACY OF A NOVEL ANTIMICROBIAL SOLUTION AGAINST PHYTOPATHOGENIC BACTERIA, AND FEASIBILITY AS A VEGETABLE SEED TREATMENT” filed on March 23, 2010, the entireties of which are hereby incorporated by reference.
  • the present disclosure is generally related to compositions and methods of use thereof for reducing the microbial load of plant seeds and sprouts.
  • plants may become infected with phytopathogenic bacteria that may eventually contaminate the seeds.
  • the mechanisms by which seeds can become infested by bacteria vary significantly based on the plant species and how the seeds are produced.
  • the location of the bacterium in the seed may also vary.
  • bacteria may be on the seed coat, but it is also possible for the bacteria to reside deep within the seed.
  • the location of the bacterium in or on the seeds significantly influences the likelihood of successful seed treatments. Infested seeds are virtually indistinguishable from non-infested seeds, and as a result it is difficult to physically segregate them.
  • plant pathogenic bacteria will not negatively affect seed qualities like storability, germination or vigor; however, once planted, infested seeds will give rise to infected seedlings and to diseases affecting the stem, leaves and/or flowers. As the resulting seedlings will be infected early in the plant cycle, the likelihood of a disease outbreak and economic losses is high
  • phytopathogenic fungi may, in addition to give rise to infected seedlings, also impact the germination potential of seeds, leading to reduced productivity of commercial seed batches. Seed contamination by phytopathogens is a cause of great losses in agriculture. Infection of Cucurbitaceae by, for instance, Acidovorax may be responsible for up to 80% of losses of watermelon crops worldwide. Contamination of grass seeds by fungi (e.g. , P. desiperda) and phytoplasma are also associated with losses by affecting either the germination potential of seeds or the appearance and growth of sprouts and leaves. Besides phytobacterial colonization of seeds and plant crops, microbial populations can include pathogens likely to cause foodbome illnesses. For more than a decade, alfalfa sprouts have been associated with outbreaks of foodbome illness (Safranek ef al., (2009) MMWR 58: 1 -
  • Salmonellosis was associated with consumption of alfalfa sprouts in two large outbreaks in 1994 in Sweden and Finland (Ponka et al., (1995) Lancet 345: 462-463). Salmonella bovismorbificans was the causative agent and the vehicle was Australian alfalfa seeds which had received an antimicrobial treatment of 0.5% sodium hypochlorite for 45 mins. S.
  • bovismorbificans was also isolated from germinated sprouts, but not the seeds. Since 1995, raw and lightly cooked sprouts have been implicated in outbreaks of foodbome illness in the United States, mostly involving alfalfa sprouts, and to a lesser extent cress, mung bean, and clover sprouts. Thirteen Salmonella serotypes were isolated from the clinical cases. Examples include S. Newport associated with alfalfa sprouts (Oregon Health Division (1995) Oregon & BC. Communicable Dis. Summary), S. Stanley from alfalfa sprouts (Mohon ef al. , (1997) J. Infect. Dis. 175: 876-882), and S. Senftenberg from an alfalfa and clover sprout mixture.
  • Seeds are generally recognized as sources of sprout-related disease outbreaks and pathogens including Salmonella have been isolated from naturally-contaminated seeds (Inami & Moler (1999) J. Food Prot. 62: 662-664; Mukherjee et al. , (2007) Intern. J. Food Microbiol. 120: 296-302; Taormina & Beuchat (1999) J. Food Prot. 62: 850-856).
  • Various treatments, including chlorine-based disinfectants have been tested to identify disinfection procedures that eliminate pathogens while preserving germinability of the seeds (see, for example: Jaquette ef al., (1996) Appl. Environ. Microbiol. 62: 2212-2215; Kumar ef al. , (2006) J. Food Prot.
  • Taormina & Beuchat ((1999) J. Food Prot. 62: 318-324) determined the efficacy of various chemicals, including calcium hypochlorite, USS-1400 (acidified CI0 2 ), acidified sodium hypochlorite, sodium chlorite, hydrogen peroxide, ethyl alcohol, trisodium phosphate,
  • TSUNAMI .RTM, VORTEXX.RTM and VEGI-CLEAN.RTM a citrus-citric-based product for the elimination of E. coli 0157:1-17 from alfalfa seeds and their survivability of the pathogen on seeds stored for prolonged periods at different temperatures.
  • the results did not clearly demonstrate that any of these chemicals as individual treatments, or in combination with heat and chemical treatment, would ensure elimination of E. coli 0157:H7 from alfalfa seeds.
  • the present disclosure encompasses embodiments of methods of reducing the microbial load of a vegetable seed or a sprout thereof, comprising contacting the vegetable seed or the sprout thereof, with a composition comprising levulinic acid and a detergent for a period suitable for reducing a microbial population of the vegetable seed or the sprout thereof.
  • the detergent can be sodium dodecyl sulfate (SDS).
  • the concentration of levulinic acid in the composition is between about 0.1 % wt/vol and about 5% wt/vol levulinic acid.
  • the concentration of levulinic acid in the composition is about 3% levulinic acid.
  • the concentration of SDS in the composition is between about 0.01 % wt/vol and about 0.05% wt/vol SDS.
  • the concentration of SDS in the composition is about 0.05% SDS.
  • the composition comprises about 3% wt/vol levulinic acid and about 0.05% wt/vol SDS.
  • the step of contacting the vegetable seed can comprise immersing the vegetable seed or the sprout thereof in the composition.
  • the step of contacting the vegetable seed or the sprout thereof, with the composition can comprise contacting the seed with the composition and applying a vacuum thereto, whereby air from the vegetable seed or the sprout thereof, can be replaced with a volume of the composition.
  • the composition reduces a bacterial population located on the exterior surface of the seed coat (testa) of a vegetable seed, located in or on the seed contents beneath the testa, or located on and beneath the testa of the vegetable seed.
  • the composition can reduce a microbial population located on the exterior surface of the seed coat (testa) of a vegetable seed, located in or on the seed contents beneath the testa, or located on and beneath the testa of the vegetable seed, wherein the microbial population is a bacterial population.
  • Fig. 1 is a graph illustrating the effects upon the germination of watermelon seeds of washes having 3% levulinic acid and 0.05% SDS. Seeds were treated by either washing or washing and vacuum-induced impregnation of the seeds. The control was washing the only with 0.05% SDS.
  • Fig. 2 is a graph illustrating the reduction in microbial colonization of watermelon seeds by Acidovorax avenae subsp. citrulli using 3% levulinic acid and 0.05% SDS applied to the seeds as a wash or as a wash combined with vacuum-induced impregnation.
  • Figs. 3A and 3B are a pair of graphs illustrating the effect of 0.5% levulinic acid on in vitro growth of Acidovorax avenae subsp. citrulli (Aac), Pantoea ananatis (PNA), Xanthomonas campestris pv. campestris (XCC), Xanthomonas campestris pv. vesicatoria (XCV) Clavibacter michiganenses subsp. michiganenses (CMM) and Erwinia carotovora subsp. carotovora (ECC).
  • Fig. 3A illustrates the growth curves in nutrient broth of the bacterial species in the absence of levulinic acid.
  • Fig. 3B illustrates the growth curves for the same bacterial species as in Fig. 3A, cultured in the presence of 0.5% levulinic acid.
  • Fig. 4 is a graph illustrating the effect of seed treatments with 3% levulinic acid
  • Fig. 5 is a graph illustrating the effect of seed treatment with 3% levulinic acid and 0.05% SDS on BFB seedling transmission over time for melon seeds naturally infested with A. avenae subsp. citrulli.
  • Fig. 6 is a graph illustrating the effect seed treatments with 3% levulinic acid on mean germination percentage for melon seedlings 14 days after planting.
  • Fig. 7 is a graph illustrating the effect of 3% levulinic acid on mean shoot length and mean root length of melon seedlings 14 days after germination.
  • a support includes a plurality of supports.
  • compositions like those disclosed herein but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc. , however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.
  • Consisting essentially of or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • microorganism or "microbe” as used herein is intended to include any bacterial species including, but not limited to, coliforms ⁇ Escherichia spp. , Salmonella spp. , and the like), Bacillus spp. and phytobacteria, or spores formed by any of these.
  • phytobacteria refers to species of bacteria that induce pathologies in plants including, but not limited to: Burkholderia ambifaria, B. andropogonis, B. anthina, B. brasilensis, B. caledonica, B. caribensis, B. caryophylli, B. cenocepacia, B. cepacia, B. cepacia complex, B. dolosa, B. fungorum, B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururiensis, B. mallei, B. multivorans, B. oklahomensis, B. phenazinium, B.
  • chrysanthemi E. mallotivora, E. papayae, E. persicina, E. psidii, E. pyrifoliae, E. rhapontici, E. toletana, E. tracheiphila, Pantoea ananatis, P. citrea, Pseudomonas syringae, Xanthomonas albilineans, X. alfalfae, X. ampelina, X. arboricola, X. axonopodis, X. boreopolis, X. badrii, X. bromi, X. campestris, X. assavae, X. citri, X. codiaei, X. cucurbitae, X. cyanopsidis, X. cynarae,
  • X. phaseoli X. pisi, X. populi, X. acchari, X. theicola, X. translucens, X. vasicola, X. vesicatoria, and the like.
  • Phytobacteria also include Phytoplasma bacteria such as Ca. Phytoplasma
  • Phytoplasma fraxini Ca. Phytoplasma japonicum, Ca. Phytoplasma luffae, Ca. Phytoplasma mali, Ca. Phytoplasma oryzae, Ca. Phytoplasma palmae, Ca. Phytoplasma phoenicium, Ca. Phytoplasma pruni, Ca. Phytoplasma prunorum, Ca. Phytoplasma pyri, Ca. Phytoplasma rhamni, Ca. Phytoplasma solani, Ca. Phytoplasma spartii, Ca. Phytoplasma trifolii, Ca.
  • microorganism as used herein also includes fungi such as (but not limited to) those of the Pyrenophora (Pyrenophora semeniperda, P. teres, P. graminea and P. tritici- repentis), Bipolaris (B. cactivora, B. cookie, B. incurvata, B. sacchari) and Cochliobolus (C. carbonum, C. heterostrophum, C. lunatus, C. stenospilus), Erysiphe genera and the like.
  • Pyrenophora Pyrenophora semeniperda, P. teres, P. graminea and P. tritici- repentis
  • Bipolaris B. cactivora
  • B. cookie B. incurvata
  • B. sacchari B. sacchari
  • Cochliobolus C. carbonum, C. heterostrophum, C. lunatus, C. stenospilus
  • antimicrobial refers to a compound that exhibits microbicidal or microbiostatic properties that enable the compound to kill, destroy, inactivate, or neutralize a microorganism; or to prevent or reduce the growth, ability to survive, or propagation of a microorganism.
  • microbial load refers to a population of microbial organisms colonizing a vegetable seed or a sprout thereof.
  • the microbial load is not limited to
  • phytobacteria that may or may not be harmful to the colonized seed or sprout thereof, but may also include opportunistic colonizers that reside on or in a seed or a sprout thereof as a result of mere contact with a population of such organisms.
  • the term "acid” as used herein refers to any chemical compound that, when dissolved in water, gives a solution with a hydrogen ion activity greater than in pure water, i.e. a pH less than 7.0.
  • An "organic acid” is a carbon-containing compound (except for carbonic acid) with acidic properties.
  • a monoprotic acid is an acid that is able to donate one proton per molecule during ionization.
  • an anti-microbial composition refers to a concentration of active agent that provides the desired effect, i.e., log order reduction in surface microbial counts without reducing the viability of the treated vegetable seed or the sprout thereof, or the ability of the seed to germinate.
  • organoleptic properties refers to properties that can be detected by human or animal senses (taste, color, odor, feel) unaided by mechanical and analytical devices.
  • the present disclosure encompasses methods of using antimicrobial compositions comprising levulinic acid and a detergent for reducing the microbial load of a seed or a germinated sprout thereof.
  • the methods of the present disclosure are useful for reducing the microbial load of seeds or seedlings colonized or infected with phytobacteria.
  • the wash methods of the disclosure are effective in significantly decreasing microbial populations resident on or within seeds and seedlings, when the microbial populations are adventitious colonizations by bacteria potentially pathogenic to a consumer.
  • the seeds or the parent plants may have been irrigated with water contaminated by fecal organisms including, but not limited to, E. coli, S. typhimurium, and the like.
  • Such bacteria can attach to developing seeds or seedlings, proliferate or merely reside thereon and thereby constitute a threat of digestive tract illnesses to the consumer of food derived from such plant products.
  • the data of the present disclosure supports the efficacy of the methods to decrease seed- and seedling-borne microbial populations while not significantly decreasing the viability of the treated material. Thus propagation of the seeds is not adversely affected, their health can be improved, providing economic and safety benefits.
  • Acidovorax avenae subsp. citrulli the causal agent of bacterial fruit blotch of cucurbits (melon, watermelon, pumpkins, etc.), and several other phytobacteria, including Xanthomonas campestris pv. campestris (black rot of crucifiers), Xanthomonas vesicatoria (bacterial spot of pepper), Pantoea ananatis (center rot of onion), Erwinia carotovora subsp. carotovora (Erwinia soft rot), and Clavibacter michiganensis subsp.
  • michiganesis bacterial canker of tomato
  • the efficacy of the wash as a seed treatment was also evaluated and assessed with respect to its impact on fruit and seed quality, the germination of the seeds, and the health and viability of the resulting sprouts and seedlings.
  • levulinic acid killed the pathogens, i.e. reduced the number of viable cells in the liquid broth. Broth cultures of all the bacteria were plated after the incubation period and showed that the action of the levulinic acid was Dactenciaai rather than bacteristatic. Each bacterium was evaluated 1 U times and the experiment was repeated.
  • levulinic acid As a seed treatment, 3% levulinic acid and 0.05% SDS was applied as a soak or a vacuum treatment (both for 1 hr) to 4 replicates of 100 watermelon seeds naturally infected with Acidovorax avenae subsp. citrulli. Seeds treated with 0.05% SDS served as a negative control. Germination percentages, as shown in Fig. 1 , were 78% and 84% for seeds that were vacuum-infiltrated or soaked in levulinic acid, respectively, and there was no statistically significant difference in germination between levulinic acid-treated seeds and those treated with 0.05% SDS (89.25%). However, seedling disease transmission, as shown in Fig.
  • hypochlorite, calcium hypochlorite, acidified sodium chlorite, acidified chlorine dioxide, sodium phosphate, peroxyacetic acid, and hydrogen peroxide have been evaluated to reduce of E. coli 0157:H7 contamination on alfalfa seeds.
  • none of the above chemical treatments has been shown to eliminate E. coli 0157:H7 on alfalfa seeds and sprouts.
  • Alfalfa seeds were inoculated with a 5-strain mixture of E. coli 0157:H7 or S. typhimurium DT 104 at 10 8 CFU/g and then dried at 21 °C in a laminar hood for up to 72 hr.
  • E. coli 0157:H7 counts at 4 hr, 24 hr, 48 hr, and 72 hr of drying were about 8.1 , about 4.8, about 4.0, and about 4.0 log-i 0 CFU/g, respectively; and S. typhimurium DT 104 counts were about 6.6, about 4.4, about 4.3, and about 4.1 log 10 CFU/g, respectively.
  • E. coli 0157:H7 and S. typhimurium DT 104 populations by about 5.6, greater than 3.3, greater than 2.4, greater than 2.3; and about 6.4, greater than 2.7, about 4.3, about 2.4 log 10 CFU/g on seeds dried for 4 hr, 24 hr, 48 hr, and 72 hr, respectively.
  • Seeds contaminated with 10 4 E. coli 0157:H7/g, dried for 2 hr at 21 °C, and treated with 0.5% levulinic acid plus 0.05% SDS at 40°C for up to 5 min were E. coli 0157:H7-negative by direct plating (less than five E. coli 0157:H7/g) but were positive by enrichment culture.
  • levulinic acid is an effective antimicrobial compound against a range of gram-negative and gram-positive plant pathogenic bacteria. Even at 0.5% concentration, the solution limited in vitro population growth for all pathogens tested. Seed treatment with 3% levulinic acid suppressed the seed-to-seedling transmission of BFB on , resort. , . c
  • E. coli 0157 H7 populations were reduced by about 3 log CFU/g by drying at 21 °C for
  • S. typhimurium DT 104 populations on alfalfa seeds after drying in a laminar flow hood for 4 hr were consistently from about 10 6 to about 10 7 CFU/g after treatment with PBS.
  • the treatment system may be a contributing factor to the pathogens surviving the antimicrobial treatments.
  • the seeds in 5 to 15-lb lots in a nylon bag were not all fully exposed to the treatment solutions, thereby resulting in survivors.
  • a treatment process that ensures each seed receives exposure to the treatment solution for at least 5 mins is contemplated to increase treatment efficacy.
  • the present disclosure therefore, encompasses embodiments of a method of reducing the microbial load of a vegetable seed or a sprout thereof, comprising contacting the vegetable seed or the sprout thereof, with a composition comprising levulinic acid and a detergent for a period suitable for reducing a microbial population of the vegetable seed or the sprout thereof.
  • the detergent can be sodium dodecyl sulfate (SDS).
  • the concentration of levulinic acid in the composition is between about 0.1 % wt/vol and about 5% wt/vol levulinic acid.
  • the concentration of levulinic acid in the composition is about 3% levulinic acid.
  • the concentration of SDS in the composition is between about 0.01 % wt/vol and about 0.5% wt/vol SDS.
  • the concentration of SDS in the composition is about 0.05% SDS.
  • the composition comprises about
  • the step of contacting the vegetable seed can comprise immersing the vegetable seed or the sprout thereof in the composition.
  • the step of contacting tne vegetaoie seed or the sprout thereof, with the composition can comprise contacting the seed with the composition and applying a vacuum thereto, whereby air from the vegetable seed or the sprout thereof, can be replaced with a volume of the composition.
  • the composition can reduce a bacterial population located on the exterior surface of the seed coat (testa) of a vegetable seed, located in or on the seed contents beneath the testa, or located on and beneath the testa of the vegetable seed.
  • the composition can reduce a microbial population located on the exterior surface of the seed coat (testa) of a vegetable seed, located in or on the seed contents beneath the testa, or located on and beneath the testa of the vegetable seed, wherein the microbial population is a bacterial population.
  • ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited .
  • a concentration range of "about 0.1 % to about 5%" should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g.
  • ⁇ ne term "about” can include ⁇ 1 %, ⁇ 2%, ⁇ 3%, ⁇ 4%, ⁇ 5%, ⁇ 6%, ⁇ 7%, ⁇ 8%, ⁇ 9%, or ⁇ 10%, or more of the numerical value(s) being modified.
  • Bacterial strains Five isolates of Escherichia coli 0157:H7, including strain 932 (human isolate), E009 (beef isolate), E0018 (cattle isolate), E0122 (cattle isolate), E0139 (deer jerky isolate); and five isolates of Salmonella typhimurium DT104, including strain H2662 (cattle isolate), 1 942A (cattle isolate), 13068A (cattle isolate), 152N17-1 (dairy isolate) and H3279 (human isolate) were used. Nalidixic acid-resistant (50pg/ml) E. coli 0157:H7 were used to facilitate
  • Each E. coli 0157: H7 and Salmonella strain was grown in tryptic soy broth (TSB, Beckon Dickinson, Sparks, MD) at 37°C for 18 hr. Approximately equal cell numbers of each of the five strains were combined and used as a 5-strain mixture with cell numbers being adjusted by spectrophotometric (Spectronic Instruments, Rochester, NY) determination as described in Zhao ef a/. , (2009) J. Food Prot. 72: 928-936, incorporated herein by reference in its entirety.
  • Bacterial cell numbers were confirmed by plating 0.1 ml of serial dilutions (1 : 10) in 0.1 % peptone on tryptic soy agar (TSA, Beckon Dickinson) and Sorbitol MacConkey agar (SMA, Oxoid, Hampshire, UK) for E. coli 0157: 1-17, and TSA and XLD agar (Beckon Dickinson) for Salmonella and incubating all plates at 37°C for 24 hr, after which colonies were counted.
  • TSA tryptic soy agar
  • SMA Sorbitol MacConkey agar
  • SMA Oxoid, Hampshire, UK
  • Alfalfa seeds were obtained from Caudill Seeds Co. (Louisville, KY), and had a germination percentage of approximately 91 %. Before each trial, 25ml of sterile tap water and 25g of alfalfa seeds were added to a WHI RL-PAK.RTM bag (Nasco, Fort).
  • typhimurium DT 104 (high inocula at 10 8 -10 9 CFU/ml and low inocula at 10 3 - 10 4 CFU/ml) was inoculated on the surface of the seeds in each glass beaker under a laminar flow hood. A sterile plastic spoon was used to mix for 2 min the seeds with bacterial suspension. The glass beakers were held in a laminar flow hood for 1 hr, 4 hr, 24 hr, 48 hr, and
  • DT104 (10 8 -10 9 CFU/ml or 10 3 -10 4 CFU/ml).
  • a sterilize plastic spoon was used to mix for 2 min the seeds with each bacterial suspension.
  • the seeds were held in the laminar flow hood for 1 hr with intermittent mixing by plastic spoon before being placed into a nylon bag (85 cm x 40 cm) immediately before receiving the levulinic acid plus SDS or calcium hypochlorite treatment.
  • Inactivation of E. coli 0157:H7 and Salmonella on alfalfa seeds Inoculated, dried alfalfa seeds (50g samples) were placed in a 1000 ml glass beaker containing 200 ml of treatment solution and agitated by a magnetic stir bar at 50 ppm at 21 °C for 0, 1 , 2, 5, 10, 20, 30, and 60 min. Following treatment, 25g of seeds was placed in a stomacher bag containing 25 ml 0.1 M phosphate buffer, pH 7.2 (PBS) for levulinic acid plus SDS-treated seeds or neutralizing buffer (50x) for calcium hypochlorite-treated seeds and pummeled for 1 min.
  • PBS phosphate buffer, pH 7.2
  • 50x neutralizing buffer
  • the washing container consisted of a stainless steel bin measuring 6 x 6 x 6 ft with an external pump delivering filtered air. On the bottom of the bin, pipes were fitted to deliver air into the liquid sanitizer by an air pump.
  • the washing apparatus generally held 302 liters of solution and can wash seeds 4 to 6 nylon bags and each bag can hold 6.8 kg of seeds.
  • Treated and non-treated seeds (5g or number of seeds counted per replicate) were placed on the surface of a plastic tray. Three plastic trays were stacked vertically and top tray was filled with 100 ml of sterile deionized water or tap water which fed to the lower trays to maintain uniform moisture content for the seeds. The seeds were incubated at 22°C for 72 hr. Each evaluation was based on three replicates of 200-400 seeds each. A seed was considered to have germinated if the seed coat was broken and a visible sprout was extending at least 8 mm from the original seed. Swollen seeds or ruptured seeds with sprout tissue still inside were not counted as germinated, because such seeds rarely sprouted completely after an additional 24 hr of incubation.
  • E. coli 01 57: H7 on alfalfa seeds after inoculation and dried in a laminar hood for 4 h r was consistently at 10 8 CFU/g for PBS-treated seeds. Seeds treated with 20,000 ppm calcium hypochlorite or 0.5% levulinic acid plus 0.05% SDS for up to 60 min reduced E. coli 01 57:1-17 populations by greater than 6 and 5 log CFU/g, respectively.
  • the population of E. coli 0157: 1-17 was reduced by about 3 log CFU/g during drying for
  • Drying inoculated Salmonella on alfalfa seeds for 24 hr, 48 hr, or 72 hr decreased the population of Salmonella by ca. 2 log CFU/g.
  • Both treatments of 20,000 ppm calcium hypochlorite and 0.5% levulinic acid plus 0.05% SDS for 5 min reduced Salmonella to levels only detectable by selective enrichment culture (Table 2). . . . ⁇ ⁇ n , , u u . ⁇ l t . ...
  • Levulinic acid prevented in vitro growth of all phytopathogenic bacteria screened, as shown in Figs. 3A and . , . .. . . . ,. , réelle . , . . . , on. in me aDsence of levulinic acid, populations of all six bacteria increased over time, with G. michiganensis subsp. michiganensis increasing the least. In contrast, at concentrations as low as 0.5 %, levulinic acid prevented the growth of all bacteria, including the gram-positive species
  • samples were planted on two layers of blotter paper (Hoffman Manufacturing Inc.) saturated with sterile water in transparent germination boxes. Seeds were incubated under conditions of 30°C, 100% RH and continuous fluorescent light for 14 days. Each day after planting, seedlings were visually examined and the proportion of seedlings that germinated and developed typical BFB symptoms was enumerated 14 days after planting. This experiment was repeated twice.
  • blotter paper Hoffman Manufacturing Inc.

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  • Pretreatment Of Seeds And Plants (AREA)

Abstract

Conformément à des modes de réalisation, la présente invention porte sur un procédé de réduction de la charge microbienne d'une graine végétale ou d'une pousse de celle-ci, lequel procédé comprend la mise en contact de la graine végétale ou de la pousse de celle-ci avec une composition comprenant de l'acide lévulinique et un détergent pendant une période de temps appropriée pour la réduction d'une population bactérienne de la graine végétale ou de la pousse de celle-ci. Dans des modes de réalisation des procédés de l'invention, la composition peut réduire une population bactérienne située sur la surface extérieure du tégument (testa) d'une graine végétale, située dans ou sur le contenu de la graine au-dessous du testa ou située sur et au-dessous du testa de la graine végétale.
PCT/US2011/027719 2010-03-17 2011-03-09 Traitement antimicrobien pour des graines et des pousses WO2011115798A2 (fr)

Priority Applications (1)

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US13/510,724 US20130005811A1 (en) 2010-03-23 2011-03-09 Antimicrobial treatment for seeds and sprouts

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US31492210P 2010-03-17 2010-03-17
US61/314,922 2010-03-17
US31658210P 2010-03-23 2010-03-23
US61/316,582 2010-03-23

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WO2011115798A2 true WO2011115798A2 (fr) 2011-09-22
WO2011115798A3 WO2011115798A3 (fr) 2012-01-05

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160174557A1 (en) * 2013-07-04 2016-06-23 Vilmorin & Cie Treatment for seeds disinfection
EP3110241A4 (fr) * 2014-02-24 2017-08-16 Advent Seed Processing Ltd. Méthode de désinfection interne de graines en combinant la prégermination de graine avec l'infiltration sous vide
US20210378163A1 (en) * 2018-11-28 2021-12-09 Agri-Neo Inc. Method for controlling pathogens and/or preventing diseases resulting from the presence of the pathogens in and/or on seeds
RU2794356C1 (ru) * 2022-08-19 2023-04-17 Федеральное государственное бюджетное научное учреждение Уфимский федеральный исследовательский центр Российской академии наук Композиция с фунгицидной и росторегулирующей активностью для предпосевной обработки семян зерновых культур
WO2024155782A1 (fr) * 2023-01-19 2024-07-25 Kannar Earth Science, Ltd. Compositions et procédés pour lutter contre des agents pathogènes de plantes

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4372975B2 (ja) * 2000-06-22 2009-11-25 株式会社テイエス植物研究所 種子病害防除方法
AU2002244133A1 (en) * 2001-02-20 2002-09-04 Paul Stamets Delivery systems for mycotechnologies, mycofiltration and mycoremediation
TW200733880A (en) * 2005-09-09 2007-09-16 Suntory Ltd Method for low light cultivation and plant growth-promoting agent
WO2008126374A1 (fr) * 2007-03-30 2008-10-23 Cosmo Oil Co., Ltd. Agent permettant d'améliorer la résistance aux alcalins d'une plante et procédé d'amélioration de la résistance aux alcalins d'une plante
WO2009030641A1 (fr) * 2007-09-06 2009-03-12 Vitec Speciality Chemicals Limited Compositions d'organosilane antimicrobiennes stabilisées dans l'eau et procédés d'utilisation

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160174557A1 (en) * 2013-07-04 2016-06-23 Vilmorin & Cie Treatment for seeds disinfection
US12035716B2 (en) * 2013-07-04 2024-07-16 Vilmorin & Cie Treatment for seeds disinfection
EP3110241A4 (fr) * 2014-02-24 2017-08-16 Advent Seed Processing Ltd. Méthode de désinfection interne de graines en combinant la prégermination de graine avec l'infiltration sous vide
US20210378163A1 (en) * 2018-11-28 2021-12-09 Agri-Neo Inc. Method for controlling pathogens and/or preventing diseases resulting from the presence of the pathogens in and/or on seeds
RU2794356C1 (ru) * 2022-08-19 2023-04-17 Федеральное государственное бюджетное научное учреждение Уфимский федеральный исследовательский центр Российской академии наук Композиция с фунгицидной и росторегулирующей активностью для предпосевной обработки семян зерновых культур
WO2024155782A1 (fr) * 2023-01-19 2024-07-25 Kannar Earth Science, Ltd. Compositions et procédés pour lutter contre des agents pathogènes de plantes

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