WO2024088886A1 - Use for delaying greening and/or formation of solanine in potatoes - Google Patents

Use for delaying greening and/or formation of solanine in potatoes Download PDF

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Publication number
WO2024088886A1
WO2024088886A1 PCT/EP2023/079209 EP2023079209W WO2024088886A1 WO 2024088886 A1 WO2024088886 A1 WO 2024088886A1 EP 2023079209 W EP2023079209 W EP 2023079209W WO 2024088886 A1 WO2024088886 A1 WO 2024088886A1
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strain
bacillus
cfu
tubers
bacillus subtilis
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PCT/EP2023/079209
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French (fr)
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Albert Schirring
Michael KUBICKI
Jolanda Maud WIJSMULLER
Sander Henk Jan UWLAND
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Bayer Aktiengesellschaft
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Publication of WO2024088886A1 publication Critical patent/WO2024088886A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • 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
    • A01N63/22—Bacillus

Definitions

  • Tuber greening is regularly observed when certain environmental factors such as temperature and light are not correctly set during storage of potatoes. This is not only a cosmetic problem that is not appreciated by consumers. However, greening is also associated with the presence of higher quantities of colorless glycoalkaloids such as mainly a-solanine and also a-chaconine. Such glycoalkaloids impair the flavor by causing bitterness and could be toxic for the consumer in concentrations above 200 mg per kilo of fresh weight (for a review see Dhalsamant et al. (2022); Journal of Agricultural and Food Chemistry; available at https://doi.org/10.1021/acs.jafc.2c01169). Accordingly, there is an urgent need to reduce or delay the greening process and content of glycoalkaloids in tubers independent of storage conditions.
  • colorless glycoalkaloids such as mainly a-solanine and also a-chaconine.
  • Such glycoalkaloids impair the flavor by causing bitterness and could be toxic for the consumer in concentrations above 200 mg per
  • the present invention relates to a method of delaying and/or reducing greening and/or formation of solanine in and/or for prolonging storage stability of potatoes comprising applying prior to or at the time of planting an effective amount of a Bacillus strain to potato tubers, tuber pieces, potato transplants, apical cuttings or botanical potato seeds, planting said tubers, tuber pieces, potato transplants, apical cuttings or botanical seeds into the soil to let plants grow and obtaining potato tubers from said plants.
  • the invention also relates to the use of Bacillus subtilis sp. strain QST713 to delay and/or reduce greening and/or formation of solanine in and/or for prolonging storage stability of potatoes.
  • the genus Bacillus as used herein refers to a genus of Gram-positive, rod-shaped bacteria which are members of the division Firmicutes. Bacillus bacteria may be characterized and identified based on the nucleotide sequence of their 16S rRNA or a fragment thereof (e.g., approximately a 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, or 1500 nt fragment of 16S rRNA or rDNA nucleotide sequence).
  • the Bacillus strain of the present invention may be any one of B. acidiceler, B. acidicola, B. acidiproducens, B. aeolius, B. aerius, B. aerophilus, B.
  • benzoevorans B. beveridgei, B. bogoriensis, B. boroniphilus, B. butanolivorans, B. canaveralius, B. carboniphilus, B. cecembensis, B. cellulosilyiicus, B. cereus, B. chagannorensis, B. chungangensis, B. cibi, B. circulans, B. clarkii, B. clausii, B. coagulans, B. coaheldnsis, B. cohnii, B. decisifrondis, B. decolorationis, B. drentensis, B. farraginis, B. faslidiosus, B.
  • B. flexus firmus
  • B. foraminis B. fordii, B. fords, B. fumarioli
  • B. funiculus B. galactosidilyticus
  • B. galliciensis B. gelatini, B. gibsonii, B. ginsengi, B. ginsengihumi, B. graminis, B. halmapalus, B. halochares, B. halodurans, B. hemicellulosilyticus, B. herbertsteinensis, B. horikoshi, B. horneckiae, B. horti, B. humi, B. hwajinpoensis, B.
  • soli B. solisalsi, B. sonorensis, B. sporothermodurans, B. stratosphericus, B. subterraneus, B. subtilis, B. taecinsis, B. tequilensis, B. thermantarcticus, B. thermoamylovorans, B. thermocloacae, B. thermolactis, B. thioparans, B. thuringiensis, B. tripoxylicola, B. tusciae, B. vallismortis, B. vedderi, B. vietnamensis, B. vireti, B. wakoensis, B. weihenstephanensis, B. xiaoxiensis, and mixtures or blends thereof.
  • Suitable Bacillus strains are preferably strains of the species Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus subtilis var. amyloliquefaciens, Bacillus pumilus, or a combination thereof. It is understood that in the course of time, certain Bacillus strains may be re-classified based on the latest findings with regard to genomic similarity and/or evolutionary development. Such re-classified strain are still encompassed in the present invention. More preferably, the Bacillus strain is selected from the group consisting of Bacillus subtilis var. amyloliquefaciens strain FZB24 (available from Novozymes Biologicals Inc.
  • Bacillus amyloliquefaciens strain FZB42 available as RHIZOVITAL® from Belchim
  • Bacillus amyloliquefaciens strain D747 available as BACSTAR® from Etec Crop Solutions, NZ and also available as DOUBLE NICKEL® from Certis, US
  • Bacillus amyloliquefaciens strain ATB-BAS-010 available as Rhizofert from Artechno, Gembloux
  • Bacillus subtilis strain Y1336 available as BIOBAC® WP from Bion-Tech, Taiwan, registered as a biological fungicide in Taiwan under Registration Nos.
  • Bacillus subtilis strain MBI 600 available as Integral Pro from BASF DE
  • Bacillus subtilis strain QST713 (Accession No. NRRL B- 21661)
  • Bacillus subtilis AQ30002 (Accession No. NRRL B-50421)
  • Bacillus subtilis AQ30004 (Accession No. NRRL B-50455)
  • Bacillus pumilus QST2808 (Accession No. NRRL B-30087), mutants thereof with all the identifying characteristics of the respective strain, and combinations thereof.
  • Bacillus strain is Bacillus subtilis strain QST713 (Accession No.
  • Bacillus subtilis strain QST713 has been deposited as Accession No. NRRL B-21661. Bacillus subtilis QST713, its mutants, its supernatants, and its lipopeptide metabolites, and methods fortheir use to control plant pathogens and insects are fully described in U.S. Patent Nos. 6,060,051; 6,103,228; 6,291,426; 6,417,163 and 6,638,910. In these patents, the strain is referred to as AQ713, which is synonymous with QST713. Any references in this specification to QST713 refer to Bacillus subtilis QST713.
  • Bacillus subtilis QST713 e.g., Bacillus subtilis AQ30002 and AQ30004, deposited as Accession Numbers NRRL B-50421 and NRRL B-50455
  • Bacillus subtilis QST713 e.g., Bacillus subtilis AQ30002 and AQ30004, deposited as Accession Numbers NRRL B-50421 and NRRL B-50455
  • NRRL B-50421 deposited as Accession Numbers NRRL B-50421 and NRRL B-50455
  • subtilis 168 are 85% or greater identical to proteins in QST713.
  • a pesticidal product based on B. subtilis strain FZB24 which is as closely related to QST713 as FZB42, is classified in documents of the U.S. EPA as B. subtilis var. amyloliquefaciens . Due to these complexities in nomenclature, this particular Bacillus species is variously designated, depending on the document, as B. subtilis, B. amyloliquefaciens, and B. subtilis var.
  • the SERENADE® product (U.S. EPA Registration No. 69592-12) contains a patented strain of Bacillus subtilis (strain QST713) and many different lipopeptides that work in concert to destroy disease pathogens and provide superior antimicrobial activity.
  • the SERENADE® product is used to protect plants such as vegetables, potatoes, fruit, nut, and vine crops against diseases such as Fire Blight, Botrytis, Sour Rot, Rust, Sclerotinia, Rhizoctonia, Powdery Mildew, Bacterial Spot and White Mold.
  • the SERENADE® products are available as either liquid or dry formulations which can be applied as a foliar and/or soil treatment. Copies of U.S.
  • SERENADE® ASO SERENADE® MAX
  • SERENADE SOIL® SERENADE SOIL®
  • SERENADE® ASO (Aqueous Suspension-Organic) contains 1.34% of dried QST713 as an active ingredient and 98.66% of other ingredients. SERENADE® ASO is formulated to contain a minimum of 1 x 10 9 cfu/g of QST713 while the maximum amount of QST713 has been determined to be 3.3 x IO 10 cfu/g. Alternate commercial names for SERENADE® ASO include SERENADE BIOFUNGICIDE®, SERENADE SOIL® and SERENADE® GARDEN DISEASE. For further information, see the U.S. EPA Master Labels for SERENADE® ASO dated January 4, 2010 and SERENADE SOIL®, each of which is incorporated by reference herein in its entirety.
  • SERENADE® MAX contains 14.6% of dried QST713 as an active ingredient and 85.4% of other ingredients. SERENADE® MAX is formulated to contain a minimum of 7.3 x 109 cfu/g of QST713 while the maximum amount of QST713 has been determined to be 7.9 x 1010 cfu/g. For further information, see the U.S. EPA Master Label for SERENADE® MAX, which is incorporated by reference herein in its entirety.
  • SERENADE® OPTIMUM (or OPTI) contains 26.2% of dried QST713 as an active ingredient and 73.8% of other ingredients.
  • SERENADE® OPTIMUM (or OPTI) is formulated to contain a minimum of 1.31 x 1010 cfu/g of QST713.
  • U.S. EPA Master Label for SERENADE® OPTIMUM (or OPTI), which is incorporated by reference herein in its entirety.
  • compositions comprising 713 or a mutant thereof can be obtained by culturing Bacillus subtilis QST713 or mutants thereof according to methods well known in the art, including by using the media and other methods described in U.S. Patent No. 6,060,051.
  • Conventional large-scale microbial culture processes include submerged fermentation, solid-state fermentation, or liquid surface culture. Towards the end of fermentation, as nutrients are depleted, Bacillus subtilis cells begin the transition from growth phase to sporulation phase, such that the final product of fermentation is largely spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of Bacillus subtilis and is generally initiated by the cell in response to nutrient limitation.
  • Fermentation is configured to obtain high levels of colony forming units of Bacillus subtilis and to promote sporulation.
  • the bacterial cells, spores and metabolites in culture media resulting from fermentation may be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential-flow filtration, depth filtration, and evaporation. Fermentation broth and broth concentrate are both referred to herein as "fermentation products.”
  • Compositions used in the present invention include fermentation products.
  • the concentrated fermentation broth is washed, for example, via a diafiltration process, to remove residual fermentation broth and metabolites.
  • the fermentation broth or broth concentrate can be dried with or without the addition of carriers using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized-bed drying, drum drying, or evaporation.
  • a mutant of FZB24 that was assigned Accession No. NRRL B-50349 by the Agricultural Research Service Culture Collection is also described in U.S. Patent Application Publication No. 2011/0230345.
  • Bacillus amyloliquefaciens FZB42 is available from ABiTEP GMBH, Germany, as the plant strengthening product RHIZOVITAL®; FZB42 is also described in European Patent Publication No.
  • mutant refers to a genetic variant derived from a Bacillus strain.
  • the mutant has all the identifying characteristics of the Bacillus strain.
  • the mutant has the ability to delay and/or reduce greening and/or formation of solanine in and/or to prolong storage stability.
  • mutants are genetic variants having a genomic sequence that has greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or preferably greater than about 99% sequence identity to the plant growth-promoting Bacillus isolate and more preferably have the ability to reduce and/or delay greening of potatoes and/or the formation of solanine at least as well as the parent QST713 strain.
  • Mutants may be obtained by treating Bacillus strain cells having the above identifying characteristics with chemicals or irradiation or by selecting spontaneous mutants from a population of such cells (such as phage resistant mutants), or by other means well known to those practiced in the art and to identify those cells which still have said identifying characteristics.
  • Targeted mutations may be introduced with CRISPR/Cas genome editing techniques.
  • Delaying greening and/or the formation of solanine refers to a delay in one or both of at least 7 days, at least 15 days, at least 1 month, at least two months, at least 4 months or at least 8 months post harvest as compared to potatoes not treated according to the invention but otherwise treated in the same way.
  • Reducing greening and/or formation of glycoalkaloids such as solanine and/or chaconine in potatoes refers to a reduction in greening of at least 40% in and/or immediately underneath and/or close to the skin (until up to about 1,5 mm distance from the skin), preferably at least 50%, more preferably at least 60 %, even more preferably at least 65 and most preferably at least 70% (all per potato fresh weight) as compared to potatoes not treated according to the invention but otherwise treated in the same way.
  • reducing the formation of glycoalkaloids such as solanine and/or chaconine refers to a reduction in the content of solanine and/or chaconine in the whole potato tuber of at least 50%, or at least 60% or at least 70%, preferably at least 75%, more preferably at least 80% and most preferably at least 85% (per potato fresh weight) as compared to potatoes not treated according to the invention but otherwise treated in the same way.
  • Comparison of greening can be effected at any point in time starting with 7 days postharvest. As apparent from the example, the effect becomes more prominent with storage time. Accordingly, it is preferred that the measurement be effected at least at day 14 post-harvest or even later.
  • the potato tuber surface holds three different tissue types, the phellem, dead cells with corcky cell walls, the phellogen and phelloderm.
  • the inner tuber tissue is based on cortical cells. Solanine synthesis takes place in the living cells of the phellogen, phelloderm and cortical cells. The highest concentrations of solanine are detected within 3 mm below the surface of the potato tuber.
  • Several suitable methods for measuring solanine content in potatoes are known see e.g. EFSA Journal 2020, 18(8), 6222 (https://doi.Org/10.2903/j.efsa.2020.6222). They include the particular HPLC7DAD/MS-based method described in leri et al. (2011; Food Chemistry 125, pp750-59).
  • Storage stability refers to the time for which potatoes can be kept without significant decrease in quality. Such a significant decrease is regularly regarded as indicated by the appearance of a certain degree of greening. As such greening is also associated with a higher content of e.g. solanine (see e.g. Pavlista, 2001, Historical Materials from University of Kansas-Lincoln Extension. Paper 88), greening is also an indicator of solanine which causes bitter taste and in high doses may be toxic. Accordingly, the prolongation of storage stability according to the present invention primarily refers to such prolongation that is caused by delayed greening potatoes, as achieved, e.g., through application of the present invention.
  • storage stability of potatoes is defined as the time until said threshold of 150 mg solanine/kg potato fresh weight is reached.
  • storage stability is given until a threshold of 130 mg/kg, more preferably until 100 mg/kg.
  • increasing storage stability of potatoes means a prolonged storage of at least 7 days, at least 15 days, at least 1 month, at least two months, at least 4 months or at least 8 months where greening or the content of glycoalkaloids is reduced as compared to potatoes not treated according to the invention.
  • solanine content As illustrated by the examples of the present application illustrate the strong potential of the application of Bacillus strains for the reduction of solanine formation and greening. It is to be noted that the final level of solanine in the tuber flesh is driven by variety. Recent new registered varieties tend to have lower solanine levels compared to older varieties. These new variety releases reflect the current breeding programs to lower solanine content.
  • the present invention also relates to the use of a Bacillus strain for prolonging storage stability of potatoes, wherein such prolongation is caused by delayed greening and/or formation of solanine.
  • An application prior to planting may be done at any time post-harvest of potato tubers, cut potato pieces, apical cuttings or seeds to be planted. Accordingly, an application could take place directly after harvest as the earliest point in time, during storage or directly prior to planting the tubers into the soil. To save work, it is also possible to apply at the time of planting, that is at the same time as the tubers, tuber pieces, potato transplants, apical cuttings or seeds are planted into the soil. Apical cuttings are rooted transplants produced from tissue culture plants (see VanderZaag et al.
  • Application may be made by a seed or tuber treatment and/or as a soil treatment and/or treatment of artificial soil substrates (e.g., rockwool, perlite, glass, and coconut fiber) or treatment of circulation water in hydroponic cultures.
  • the Bacillus strain in particular Bacillus subtilis sp. strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010, can be applied to a potato tuber or potato seed, and/or a locus on which the plant grows, such as soil.
  • the Bacillus strain in particular Bacillus subtilis sp.
  • Bacillus amyloliquefaciens strain ATB-BAS-010 can be applied by any known means, such as by spraying a solution on the soil, soil surface drench, shanked-in, injected, foamed in, dipped in, applied in-furrow, as a band application along the seeding/planting line, sprayed, coated onto the tubers and/or applied by mixture with irrigation water. Application is preferably effected by spraying in furrow or dipping, or spraying or foaming the tubers or seeds.
  • tubers are sprayed prior to planting into the soil with an effective amount of a Bacillus strain, in particular Bacillus subtilis sp. strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010.
  • a Bacillus strain in particular Bacillus subtilis sp. strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010.
  • tubers are sprayed on the planter immediately prior to planting or sprayed in furrow. In-furrow application is effected through a nozzle system on the potato planter.
  • the Bacillus strain in particular the B.
  • subtilis QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 product is suspended in water and the solution is sprayed on the soil that is used to cover the tubers (the ridge of the row).
  • the tubers are sprayed with a respective solution through nozzles on the planters that are directed to the tubers.
  • tubers are treated during storage or after storage and before planting.
  • the spinning disc technology enables for low volume treatment and is effected while unloading storage and/or preparing the tubers for planting.
  • 0,5 to 1.5 l ofsolution is applied per ton of tubers.
  • the treatment can be effected by using standard nozzles operating system with volumes ranging from 5 - 10 liter of solution per ton of tubers.
  • dipping For application by dipping, at least 50%, preferably at least 60%, more preferably at least 80% or at least 90% of the surface of the tuber or seed is wetted.
  • the dipping time is usually between 10 seconds and 5 minutes, preferably between 30 seconds and 3 minutes but may vary and be higher or lower depending on the variety and environmental conditions.
  • One single application is normally sufficient and preferred. However, under certain circumstances a second or third application may be advisable or necessary in order to obtain the desired result.
  • microorganisms and particular strains described herein are all separated from nature and grown under artificial conditions such as in shake flask cultures or through scaled-up manufacturing processes, such as in bioreactors to maximize bioactive metabolite production, for example. Growth under such conditions leads to strain "domestication.” Generally, such a “domesticated” strain differs from its counterparts found in nature in that it is cultured as a homogenous population that is not subject to the selection pressures found in the natural environment but rather to artificial selection pressures.
  • the compositions comprising a Bacillus strain are liquid formulations.
  • liquid formulations include suspension concentrations and oil dispersions.
  • the compositions are solid formulations.
  • solid formulations include freeze-dried powders and spray-dried powders.
  • Said Bacillus strain in particular Bacillus subtilis sp.
  • Strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or mutant thereof is generally applied at a rate of between 3,00E+05 colony forming units (cfu)/ha and 5,00E+10 cfu/ha when treated in furrow or between 5,00E+08 cfu/ton tubers and l,00E+l 1 cfu/ton tubers when dipped or sprayed or l,00E+5 - l,00E+8 cfu / 1000 grams of botanical seeds
  • Application rates per row meter depend on row distance and accordingly the number of rows per hectare as the application rate per hectare remains the same regardless of row distance.
  • application rates also depend on the product comprising Bacillus strain, in particular B. subtilis QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or a mutant thereof.
  • the Bacillus strain in particular Bacillus subtilis sp. Strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or mutant thereof is generally applied at a rate of between 3,00E+09 colony forming units (cfu)/ha and 5,00E+9 cfu/ha when treated as full field application prior to planting.
  • rates for the product Serenade ASO comprising B are generally applied at a rate of between 3,00E+09 colony forming units (cfu)/ha and 5,00E+9 cfu/ha when treated as full field application prior to planting.
  • sub tilts QST713 range between 3,00E+07 cfu/ha and l,00E+10 cfu/ha, preferably between 3,00E+08 cfu/ha and 8,00E+09 cfu/ha, more preferably between 3,00E+09 cfu/ha and 5,00E+09.
  • this most preferred range corresponds to between l,98E+05 and 3,30E+05 cfu/m row, for 75 cm to between 2,25E+05 and 3,75E+05 cfu/m row and for 90 cm to between 2,70E+05 and 4,50E+05 cfu/m row.
  • rates for the product Serenade ASO comprising B. subtilis QST713 range between 3,00E+10 cfu/ha and l,00E+13 cfu/ha, preferably between 3,00E+l 1 cfu/ha and 8,00E+12 cfu/ha, more preferably between 3,00E+12 cfu/ha and 5,00E+12.
  • this most preferred range corresponds to between l,98E+08 and 3,30E+08 cfu/m row, for 75 cm to between 2,25E+08 and 3,75E+08 cfu/m row and for 90 cm to between 2,70E+08 and 4,50E+08 cfu/m row.
  • QST713 For another formulation of QST713 (HiCFU) rates range between l,50E+09 cfu/ha and 3,00E+l 1 cfu/ha, preferably between l,00E+10 cfu/ha and 5,00E+10 cfu/ha, more preferably between l,50E+10 cfu/ha and 3,00E+10 when treated in furrow.
  • this most preferred range corresponds to between 9,90E+05 and l,98E+06 cfu/m row, for 75 cm to between l,13E+06 and 2,25E+06 cfu/m row and for 90 cm to between l,35E+06 and 2,70E+06 cfu/m row.
  • rates range between l,50E+12 cfu/ha and 3,00E+14 cfu/ha, preferably between l,00E+13 cfu/ha and 5,00E+13 cfu/ha, more preferably between l,50E+13 cfu/ha and 3,00E+13 when treated in furrow.
  • this most preferred range corresponds to between 9,90E+08 and l,98E+09 cfu/m row, for 75 cm to between l,13E+09 and 2,25E+09 cfu/m row and for 90 cm to between l,35E+09 and 2,70E+09 cfu/m row.
  • rates for the product Serenade ASO range between 5,00E+07 cfu/ton tubers and l,00E+10 cfu/ton tubers, preferably between l,00E+08 cfu/ton tubers and 5,00E+09 cfu/ton tubers, more preferably between 5,00E+08 cfu/ton tubers and l,00E+09 cfu/ton tubers.
  • QST713 HiCFU
  • tuber application range between 7,00E+08 cfu/ton tubers and l,50E+l 1 cfu/ton tubers, preferably between l,00E+09 cfu/ton tubers and l,00E+l 1 cfu/ton tubers, more preferably between 7,50E+09 cfu/ton tubers and l,50E+10 cfu/ton tubers.
  • rates for the product Serenade ASO range between 5,00E+10 cfu/ton tubers and l,00E+13 cfu/ton tubers, preferably between l,00E+ll cfu/ton tubers and 5,00E+12 cfu/ton tubers, more preferably between 5,00E+l 1 cfu/ton tubers and l,00E+12 cfu/ton tubers.
  • QST713 HiCFU
  • tuber application range between 7,00E+ll cfu/ton tubers and l,50E+14 cfu/ton tubers, preferably between l,00E+12 cfu/ton tubers and l,00E+14 cfu/ton tubers, more preferably between 7,50E+12 cfu/ton tubers and l,50E+13 cfu/ton tubers.
  • rates for the product Rhizofert comprising Bacillus amyloliquefaciens, ATB - BAS-010, range between 3,00E+10 cfu/ha and l,00E+13 cfu/ha, preferably between 3,00E+l 1 cfu/ha and 8,00E+12 cfu/ha, more preferably between 3,00E+12 cfu/ha and 5,00E+12.
  • this most preferred range corresponds to between l,98E+08 and 3,30E+08 cfu/m row, for 75 cm to between 2,25E+08 and 3,75E+08 cfu/m row and for 90 cm to between 2,70E+08 and 4,50E+08 cfu/m row.
  • rates for the product Rhizofert comprising Bacillus amyloliquefaciens, ATB -BAS- 010, range between 5,00E+10 cfu/ton tubers and l,00E+13 cfu/ton tubers, preferably between l,00E+l l cfu/ton tubers and 5,00E+12 cfu/ton tubers, more preferably between 5,00E+l l cfu/ton tubers and l,00E+12 cfu/ton tubers.
  • the Bacillus strain in particular B.
  • subtilis strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or a mutant thereof may also be applied to soil and/or plants in plug trays or to seedlings prior to transplanting to a different plant locus.
  • the strain When applied to the soil in contact with the plant roots, to the base of the plant, or to the soil within a specific distance around the base of the plant, including as a soil drench treatment, the strain may be applied as a single application or as multiple applications.
  • the strain may be applied at the rates set forth above for drench treatments or a rate of about 1 x 10 5 to about 1 x 10 8 cfu per gram of soil, 1 x 10 5 to about 1 x 10 7 cfu per gram of soil, 1 x 10 5 to about IxlO 6 cfu per gram of soil, 7 x 10 5 to about 1 x 10 7 cfu per gram of soil, 1 x 10 6 to about 5 x 10 6 cfu per gram of soil, or 1 x 10 5 to about 3 x 10 6 cfu per gram of soil.
  • the strain is applied as a single application at a rate of about 7 x 10 5 to about 1 x 10 7 cfu per gram of soil. In another embodiment, the strain is applied as a single application at a rate of about 1 x 10 6 to about 5 x 10 6 cfu per gram of soil. In other embodiments, the strain is applied as multiple applications at a rate of 10 about 1 x 10 5 to about 3 x 10 6 cfu per gram of soil.
  • the strains suitable for the present invention can be applied to botanical potato seeds using conventional treating techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed treaters, and drum coaters. Other methods, such as spouted beds may also be useful.
  • the seeds may be pre-sized before coating. After coating, the seeds are typically dried and then transferred to a sizing machine for sizing. Such sizing and treating procedures are known in the art.
  • the seed be in a sufficiently durable state that it incurs no damage during the treatment process.
  • the seed would be a seed that had been harvested from the field; removed from the plant; and separated from any cob, stalk, outer husk, and surrounding pulp or other nonseed plant material.
  • the seed would preferably also be biologically stable to the extent that the treatment would cause no biological damage to the seed. It is believed that the treatment can be applied to the seed at any time between harvest of the seed and sowing of the seed or during the sowing process (seed directed applications).
  • the seed may also be primed according to techniques understood by those skilled in the art either before or after the treatment.
  • Treatment could vary from a thin fdm (dressing) of the formulation containing the strains suitable in the present invention on a plant propagation material, such as a seed, where the original size and/or shape are recognizable to an intermediary state (such as a coating) and then to a thicker fdm (such as pelleting) with many layers of different materials (such as carriers, for example, clays; different formulations, such as of other active ingredients; polymers; and colorants) where the original shape and/or size of the seed is no longer recognizable.
  • the seed treatment occurs to an unsown seed.
  • the term "unsown seed” is meant to include seed at any period between the harvest of the seed and the sowing of the seed in the ground for the purpose of germination and growth of the plant. Treatment to an unsown seed is not meant to include those practices in which the active ingredient is applied to the soil but would include any application practice that would target the seed during the planting process.
  • treatment occurs before sowing of the seed so that the sown seed has been pretreated with the strains according to the present invention.
  • seed coating or seed pelleting are preferred in the treatment with the strains described herein.
  • the strains suitable in the present invention are adhered onto the surface of the seed and therefore available for pest and/or disease control and for acting in reducing greening and solanine formation in the tubers.
  • the treated seeds can be stored, handled, sown and tilled in the same manner as any other active ingredient treated seed.
  • European Cultivated Potato Database ECPD available under https://en.wikipedia.org/wiki/European_Cultivated_Potato_Database
  • EU Plant Variety Database available under https://ec.europa.eu/food/plant/plant_propagation_
  • North American cultivars can be found in the Northe America Potato Variety Inventors (https://potatoassociation.org/publications-2/north-american-potato-variety- inventory/north-american-potato-variety-inventory-c-f/).
  • said potato variety is selected from the group consisting of table, processing and starch/protein potatoes.
  • Exemplary variants include for table potatoes Allians, Alouette, Annabelle, Arizona, Amora, Aster, Belana, Bintje, Cammeo, Carolus, Connect, Desiree, Fabula, Gala, Granola, Irish Cobbler, jazzy, Lady Anna, Manitou, Melody, Mila, Muse, Nicola, Panamera, Ratte, Sagita, Shangi, Spunta, Unica, Twister and Vitabella.
  • Processing potato variants include Agate, Agria, Alpha, Atlantic, Asterix, Challenger, Fiona, Favourita, Fontane, Hansa, Hermes, Innovator, Ivory Russet, Jelly, Markies, Maris Piper, Music, Kennebec, King Edward, Lady Marie, Norland, Royal, Russet Burbank, Russet Norkotah, Ranger Russet, Shepody, Snowdon, Umatilla Russet, Vivaldi and Yukon Gold.
  • Starch potatoes include the variants Altus, Avamond, Avama, Avatar, Avito, Axion, BMC, BMC, Festien, Kardal, Kuras, Saprodi, Seresta and Supporter.
  • Bacillus subtilis sp. strain QST713 that, inter alia, is known to have fungicidal action significantly delays greening and reduces the extent of greening and the content of solanine when applied pre planting. This results in a prolonged storage stability of potato tubers. Most notably, it was observed that the effect is passed on from a tuber treated and planted to daughter tubers harvested from the plant arising from said tuber. Without wishing to be bound by any scientific theory, it is believed that Bacillus subtilis sp. strain QST713 or mutants thereof colonize the tubers and later on the relevant plant parts and are thereby passed over to offspring of the original treated tuber.
  • a sample of a Bacillus subtilis strain suitable in the present invention has been deposited with the Agricultural Research Service Culture Collection located at the National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604, U.S.A., under the Budapest Treaty on March 7, 1997, and has been assigned Accession Number NRRL B-21661.
  • QST30002 (aka AQ30002) and QST30004 (aka AQ30004) have been deposited with the Agricultural Research Service Culture Collection under the Budapest Treaty on October 5, 2010 and December 6, 2010, respectively.
  • QST30002 has been assigned Accession Number NRRL B-50421
  • QST30004 has been assigned the following Accession Number NRRL B-50455.
  • the Bacillus subtilis strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. ⁇ 1.14 and 35 U.S.C. ⁇ 122.
  • the deposits represent a substantially pure culture of the deposited Bacillus subtilis strain.
  • the deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
  • the following examples are given for purely illustrative and non-limiting purposes of the present invention.
  • Example 1 MALDI mass spectrometry imaging analysis for visualization of reducing in greening and/or formation of glycoalkaloids
  • MALDI MSI MALDI mass spectrometry imaging
  • Tubers for cryosectioning were embedded in carboxymethylcellulose sodium salt (4 - 6% in water) and frozen at -20 °C for at least 12 hours. Samples were equilibrated in a cryostat CryoStar NX70 (Thermo Scientific GmbH, Bremen, Germany) for approximately 30 min at the optimal cutting temperature (-25 - -15 °C). Histological slices (20 - 35 pm) of tubers were mounted either on microscope slides (18x18x1 mm, Paul Marienfeld GmbH & Co. KG, Lauda-Kbnigshofen, Germany) directly or using thin doublesided adhesive tape (3M, Saint Paul, USA). The samples were dried in a desiccator at room temperature for a minimum of 30 min before matrix deposition.
  • MALDI matrices were sprayed on the sample using a MALDI sprayer SunCollect (SunChromticianliche Gerate GmbH, Friedrichsdorf, Germany) and matrix flow rate, sample speed and spray intervals, optimized for each matrix and tissue. Before and after matrix deposition, the sample surfaces were checked with a digital microscope VHX-6000 (Keyence Deutschland GmbH, Neu-Isenburg, Germany). The matrix-coated target was stored in a vacuum desiccator at room temperature in the dark until analysis. According to the actual storage time of potato tubers, we chose 0, 7, 14, 21 and 28 days at room temperature for the experiment. Glycoalkaloids began to biosynthesize and increase and showed obvious distribution characteristic in potato tuber tissues.
  • Strain QST71 refers to a reduction in greening and formation of a-solanine and a-chaconine of at least 5% post-harvest, the reducing in the whole potato tuber after 7 days is at least 45%. During the storage period the effect on reducing in greening and content of a-solanine and a-chaconine is at least 70% after 14 days and at least 80% up to 28 days. Table 1. Relative reducing in greening and formation of a-solanine and a-chaconine in potato tubers of different varieties (Saprodi, Fontana and Avama) treated with Bacillus subtilis sp. Strain QST713 after storage under UV light.

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Abstract

Use for delaying greening and/or formation of solanine in potatoes The present invention relates to a method of delaying and/or reducing greening and/or formation of solanine in and/or for prolonging storage stability of potatoes comprising applying prior to or at the time of planting an effective amount of a Bacillus strain to potato tubers, tuber pieces, potato transplants, apical cuttings or botanical potato seeds, planting said tubers or seeds into the soil to let plants grow and obtaining potato tubers from said plants.

Description

Use for delaying greening and/or formation of solanine in potatoes
Tuber greening is regularly observed when certain environmental factors such as temperature and light are not correctly set during storage of potatoes. This is not only a cosmetic problem that is not appreciated by consumers. However, greening is also associated with the presence of higher quantities of colorless glycoalkaloids such as mainly a-solanine and also a-chaconine. Such glycoalkaloids impair the flavor by causing bitterness and could be toxic for the consumer in concentrations above 200 mg per kilo of fresh weight (for a review see Dhalsamant et al. (2022); Journal of Agricultural and Food Chemistry; available at https://doi.org/10.1021/acs.jafc.2c01169). Accordingly, there is an urgent need to reduce or delay the greening process and content of glycoalkaloids in tubers independent of storage conditions.
This technical problem has, at least in part, been solved by the present invention.
Accordingly, the present invention relates to a method of delaying and/or reducing greening and/or formation of solanine in and/or for prolonging storage stability of potatoes comprising applying prior to or at the time of planting an effective amount of a Bacillus strain to potato tubers, tuber pieces, potato transplants, apical cuttings or botanical potato seeds, planting said tubers, tuber pieces, potato transplants, apical cuttings or botanical seeds into the soil to let plants grow and obtaining potato tubers from said plants. The invention also relates to the use of Bacillus subtilis sp. strain QST713 to delay and/or reduce greening and/or formation of solanine in and/or for prolonging storage stability of potatoes.
The genus Bacillus as used herein refers to a genus of Gram-positive, rod-shaped bacteria which are members of the division Firmicutes. Bacillus bacteria may be characterized and identified based on the nucleotide sequence of their 16S rRNA or a fragment thereof (e.g., approximately a 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, or 1500 nt fragment of 16S rRNA or rDNA nucleotide sequence). The Bacillus strain of the present invention may be any one of B. acidiceler, B. acidicola, B. acidiproducens, B. aeolius, B. aerius, B. aerophilus, B. agaradhaerens, B. aidingensis, B. akibai, B. alcalophilus, B. algicola, B. alkalinitrilicus, B. alkalisediminis, B. alkalitelluris, B. altit dinis, B. alveayuensis, B. amyloliquefaciens, B. anthracis, B. aquimaris, B. arsenicus, B. aryabhattai, B. asahii, B. atrophaeus, B. aurantiacus, B. azotoformans, B. badius, B. barbaricus, B. bataviensis, B. beijingensis, B. benzoevorans, B. beveridgei, B. bogoriensis, B. boroniphilus, B. butanolivorans, B. canaveralius, B. carboniphilus, B. cecembensis, B. cellulosilyiicus, B. cereus, B. chagannorensis, B. chungangensis, B. cibi, B. circulans, B. clarkii, B. clausii, B. coagulans, B. coahuilensis, B. cohnii, B. decisifrondis, B. decolorationis, B. drentensis, B. farraginis, B. faslidiosus, B. firmus, B. flexus, B. foraminis, B. fordii, B. fords, B. fumarioli, B. funiculus, B. galactosidilyticus, B. galliciensis, B. gelatini, B. gibsonii, B. ginsengi, B. ginsengihumi, B. graminis, B. halmapalus, B. halochares, B. halodurans, B. hemicellulosilyticus, B. herbertsteinensis, B. horikoshi, B. horneckiae, B. horti, B. humi, B. hwajinpoensis, B. idriensis, B. indicus, B. infantis, B. infernus, B. isabeliae, B. isronensis, B. jeotgali, B. koreensis, B. korlensis, B. kribbensis, B. krul chiae, B. lehensis, B. lentus, B. licheniformis, B. litoralis, B. locisalis, B. luciferensis, B. luteolus, B. macciuensis, B. macycie, B. mannanilyticus, B. marisflcivi, B. marmcirensis, B. massiliensis, B. megaterium, B. methanolicus, B. methylotrophicus, B. mojavensis, B. muralis, B. murimartini, B. mycoides, B. nanhciiensis, B. nanhaiisediminis, B. nealsonii, B. neizhouensis, B. niabensis, B. niacini, B. novalis, B. oceanisediminis, B. odysseyi, B. okhensis, B. okuhidensis, B. oleronius, B. oshimensis, B. panciciterrcie, B. patagoniensis, B. persepolensis, B. plakortidis, B. pocheonensis, B. polygoni, B. pseudocilcaliphilus, B. pseudofirmus, B. pseudomycoides, B. psychrosaccharolyticus, B. pumilus, B. qingdaonensis, B. rigui, B. ruris, B. safensis, B. salcirius, B. saliphilus, B. schlegelii, B. selencitarsenatis, B. selenitireducens, B. seohciecinensis, B. shackletonii, B. siamensis, B. simplex, B. siralis, B. smithii, B. soli, B. solisalsi, B. sonorensis, B. sporothermodurans, B. stratosphericus, B. subterraneus, B. subtilis, B. taecinsis, B. tequilensis, B. thermantarcticus, B. thermoamylovorans, B. thermocloacae, B. thermolactis, B. thioparans, B. thuringiensis, B. tripoxylicola, B. tusciae, B. vallismortis, B. vedderi, B. vietnamensis, B. vireti, B. wakoensis, B. weihenstephanensis, B. xiaoxiensis, and mixtures or blends thereof.
Suitable Bacillus strains are preferably strains of the species Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus subtilis var. amyloliquefaciens, Bacillus pumilus, or a combination thereof. It is understood that in the course of time, certain Bacillus strains may be re-classified based on the latest findings with regard to genomic similarity and/or evolutionary development. Such re-classified strain are still encompassed in the present invention. More preferably, the Bacillus strain is selected from the group consisting of Bacillus subtilis var. amyloliquefaciens strain FZB24 (available from Novozymes Biologicals Inc. (Salem, Virginia) or Syngenta Crop Protection, LLC (Greensboro, North Carolina) as the fungicide TAEGRO® or TAEGRO® ECO (EPA Registration No. 70127-5)), Bacillus amyloliquefaciens strain FZB42 (available as RHIZOVITAL® from Belchim), Bacillus amyloliquefaciens strain D747 (available as BACSTAR® from Etec Crop Solutions, NZ and also available as DOUBLE NICKEL® from Certis, US), Bacillus amyloliquefaciens strain ATB-BAS-010 (available as Rhizofert from Artechno, Gembloux); Bacillus subtilis strain Y1336 (available as BIOBAC® WP from Bion-Tech, Taiwan, registered as a biological fungicide in Taiwan under Registration Nos. 4764, 5454, 5096 and 5277), Bacillus subtilis strain MBI 600 (available as Integral Pro from BASF DE), Bacillus subtilis strain QST713 (Accession No. NRRL B- 21661), Bacillus subtilis AQ30002 (Accession No. NRRL B-50421), Bacillus subtilis AQ30004 (Accession No. NRRL B-50455), Bacillus pumilus QST2808 (Accession No. NRRL B-30087), mutants thereof with all the identifying characteristics of the respective strain, and combinations thereof. It is most preferred that the Bacillus strain is Bacillus subtilis strain QST713 (Accession No. NRRL B-21661) or Bacillus amyloliquefaciens strain ATB-BAS-010. Bacillus subtilis strain QST713 has been deposited as Accession No. NRRL B-21661. Bacillus subtilis QST713, its mutants, its supernatants, and its lipopeptide metabolites, and methods fortheir use to control plant pathogens and insects are fully described in U.S. Patent Nos. 6,060,051; 6,103,228; 6,291,426; 6,417,163 and 6,638,910. In these patents, the strain is referred to as AQ713, which is synonymous with QST713. Any references in this specification to QST713 refer to Bacillus subtilis QST713. Particular variants of Bacillus subtilis QST713 (e.g., Bacillus subtilis AQ30002 and AQ30004, deposited as Accession Numbers NRRL B-50421 and NRRL B-50455) that would also be suitable for the present invention are described in U.S. Patent Publication No. 2012/0231951.
At the time of filing U.S. Patent Application No. 09/074,870 in 1998, which corresponds to the above patents, the strain was designated as Bacillus subtilis based on classical, physiological, biochemical and morphological methods. Taxonomy of the Bacillus species has evolved since then, especially in light of advances in genetics and sequencing technologies, such that species designation is based largely on DNA sequence rather than the methods used in 1998. After aligning protein sequences from B. amyloliquefaciens FZB42, B. subtilis 168 and QST713, approximately 95% of proteins found in B. amyloliquefaciens FZB42 are 85% or greater identical to proteins found in QST713; whereas only 35% of proteins in B. subtilis 168 are 85% or greater identical to proteins in QST713. However, even with the greater reliance on genetics, there is still taxonomic ambiguity in the relevant scientific literature and regulatory documents, reflecting the evolving understanding of Bacillus taxonomy over the past years. For example, a pesticidal product based on B. subtilis strain FZB24, which is as closely related to QST713 as FZB42, is classified in documents of the U.S. EPA as B. subtilis var. amyloliquefaciens . Due to these complexities in nomenclature, this particular Bacillus species is variously designated, depending on the document, as B. subtilis, B. amyloliquefaciens, and B. subtilis var. amyloliquefaciens . Recent efforts are in addition aimed at characterizing the present strain QST713 as belonging to B. velenzis. Therefore, we have retained the B. subtilis designation of QST713 rather than changing it to B. amyloliquefaciens or B. velenzis, as would be expected currently based solely on sequence comparison and inferred taxonomy.
The SERENADE® product (U.S. EPA Registration No. 69592-12) contains a patented strain of Bacillus subtilis (strain QST713) and many different lipopeptides that work in concert to destroy disease pathogens and provide superior antimicrobial activity. The SERENADE® product is used to protect plants such as vegetables, potatoes, fruit, nut, and vine crops against diseases such as Fire Blight, Botrytis, Sour Rot, Rust, Sclerotinia, Rhizoctonia, Powdery Mildew, Bacterial Spot and White Mold. The SERENADE® products are available as either liquid or dry formulations which can be applied as a foliar and/or soil treatment. Copies of U.S. EPA Master Labels for the SERENADE® products, including SERENADE® ASO, SERENADE® MAX, and SERENADE SOIL®, are publicly available through National Pesticide Information Retrieval System’s (NPIRS) USEPA/OPP Pesticide Product Label System (PPLS).
SERENADE® ASO (Aqueous Suspension-Organic) contains 1.34% of dried QST713 as an active ingredient and 98.66% of other ingredients. SERENADE® ASO is formulated to contain a minimum of 1 x 109 cfu/g of QST713 while the maximum amount of QST713 has been determined to be 3.3 x IO10 cfu/g. Alternate commercial names for SERENADE® ASO include SERENADE BIOFUNGICIDE®, SERENADE SOIL® and SERENADE® GARDEN DISEASE. For further information, see the U.S. EPA Master Labels for SERENADE® ASO dated January 4, 2010 and SERENADE SOIL®, each of which is incorporated by reference herein in its entirety.
SERENADE® MAX contains 14.6% of dried QST713 as an active ingredient and 85.4% of other ingredients. SERENADE® MAX is formulated to contain a minimum of 7.3 x 109 cfu/g of QST713 while the maximum amount of QST713 has been determined to be 7.9 x 1010 cfu/g. For further information, see the U.S. EPA Master Label for SERENADE® MAX, which is incorporated by reference herein in its entirety.
SERENADE® OPTIMUM (or OPTI) contains 26.2% of dried QST713 as an active ingredient and 73.8% of other ingredients. SERENADE® OPTIMUM (or OPTI) is formulated to contain a minimum of 1.31 x 1010 cfu/g of QST713. For further information, see the U.S. EPA Master Label for SERENADE® OPTIMUM (or OPTI), which is incorporated by reference herein in its entirety.
Compositions comprising 713 or a mutant thereof can be obtained by culturing Bacillus subtilis QST713 or mutants thereof according to methods well known in the art, including by using the media and other methods described in U.S. Patent No. 6,060,051. Conventional large-scale microbial culture processes include submerged fermentation, solid-state fermentation, or liquid surface culture. Towards the end of fermentation, as nutrients are depleted, Bacillus subtilis cells begin the transition from growth phase to sporulation phase, such that the final product of fermentation is largely spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of Bacillus subtilis and is generally initiated by the cell in response to nutrient limitation. Fermentation is configured to obtain high levels of colony forming units of Bacillus subtilis and to promote sporulation. The bacterial cells, spores and metabolites in culture media resulting from fermentation may be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential-flow filtration, depth filtration, and evaporation. Fermentation broth and broth concentrate are both referred to herein as "fermentation products." Compositions used in the present invention include fermentation products. In some embodiments, the concentrated fermentation broth is washed, for example, via a diafiltration process, to remove residual fermentation broth and metabolites.
The fermentation broth or broth concentrate can be dried with or without the addition of carriers using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized-bed drying, drum drying, or evaporation. A mutant of FZB24 that was assigned Accession No. NRRL B-50349 by the Agricultural Research Service Culture Collection is also described in U.S. Patent Application Publication No. 2011/0230345. Bacillus amyloliquefaciens FZB42 is available from ABiTEP GMBH, Germany, as the plant strengthening product RHIZOVITAL®; FZB42 is also described in European Patent Publication No. EP2179652, and also in Chen, et al., “Comparative Analysis of the Complete Genome Sequence of the Plant Growth-Promoting Bacterium Bacillus amyloliquefaciens FZB42,” Nature Biotechnology, Volume 25, Number 9 (September 2007). Mutants of FZB42 are described in International Publication No. WO 2012/130221, including Bacillus amyloliquefaciens ABI01, which was assigned Accession No. DSM 10-1092 by the DSMZ - German Collection of Microorganisms and Cell Cultures.
The term “mutant” refers to a genetic variant derived from a Bacillus strain. In one embodiment, the mutant has all the identifying characteristics of the Bacillus strain. In a particular instance, the mutant has the ability to delay and/or reduce greening and/or formation of solanine in and/or to prolong storage stability. In another embodiment, mutants are genetic variants having a genomic sequence that has greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or preferably greater than about 99% sequence identity to the plant growth-promoting Bacillus isolate and more preferably have the ability to reduce and/or delay greening of potatoes and/or the formation of solanine at least as well as the parent QST713 strain. Mutants may be obtained by treating Bacillus strain cells having the above identifying characteristics with chemicals or irradiation or by selecting spontaneous mutants from a population of such cells (such as phage resistant mutants), or by other means well known to those practiced in the art and to identify those cells which still have said identifying characteristics. Targeted mutations may be introduced with CRISPR/Cas genome editing techniques.
Delaying greening and/or the formation of solanine refers to a delay in one or both of at least 7 days, at least 15 days, at least 1 month, at least two months, at least 4 months or at least 8 months post harvest as compared to potatoes not treated according to the invention but otherwise treated in the same way.
Reducing greening and/or formation of glycoalkaloids such as solanine and/or chaconine in potatoes refers to a reduction in greening of at least 40% in and/or immediately underneath and/or close to the skin (until up to about 1,5 mm distance from the skin), preferably at least 50%, more preferably at least 60 %, even more preferably at least 65 and most preferably at least 70% (all per potato fresh weight) as compared to potatoes not treated according to the invention but otherwise treated in the same way. Alternatively or in addition, reducing the formation of glycoalkaloids such as solanine and/or chaconine refers to a reduction in the content of solanine and/or chaconine in the whole potato tuber of at least 50%, or at least 60% or at least 70%, preferably at least 75%, more preferably at least 80% and most preferably at least 85% (per potato fresh weight) as compared to potatoes not treated according to the invention but otherwise treated in the same way. Comparison of greening can be effected at any point in time starting with 7 days postharvest. As apparent from the example, the effect becomes more prominent with storage time. Accordingly, it is preferred that the measurement be effected at least at day 14 post-harvest or even later. The potato tuber surface, called periderm, holds three different tissue types, the phellem, dead cells with corcky cell walls, the phellogen and phelloderm. The inner tuber tissue is based on cortical cells. Solanine synthesis takes place in the living cells of the phellogen, phelloderm and cortical cells. The highest concentrations of solanine are detected within 3 mm below the surface of the potato tuber. Several suitable methods for measuring solanine content in potatoes are known see e.g. EFSA Journal 2020, 18(8), 6222 (https://doi.Org/10.2903/j.efsa.2020.6222). They include the particular HPLC7DAD/MS-based method described in leri et al. (2011; Food Chemistry 125, pp750-59).
Storage stability refers to the time for which potatoes can be kept without significant decrease in quality. Such a significant decrease is regularly regarded as indicated by the appearance of a certain degree of greening. As such greening is also associated with a higher content of e.g. solanine (see e.g. Pavlista, 2001, Historical Materials from University of Nebraska-Lincoln Extension. Paper 88), greening is also an indicator of solanine which causes bitter taste and in high doses may be toxic. Accordingly, the prolongation of storage stability according to the present invention primarily refers to such prolongation that is caused by delayed greening potatoes, as achieved, e.g., through application of the present invention. The maximum permissible value for solanine in Europe is currently set of 150 mg/kg potato fresh weight but may decrease in the future. Accordingly, storage stability of potatoes is defined as the time until said threshold of 150 mg solanine/kg potato fresh weight is reached. Preferably storage stability is given until a threshold of 130 mg/kg, more preferably until 100 mg/kg. Accordingly, increasing storage stability of potatoes means a prolonged storage of at least 7 days, at least 15 days, at least 1 month, at least two months, at least 4 months or at least 8 months where greening or the content of glycoalkaloids is reduced as compared to potatoes not treated according to the invention. Reduction in solanine content as illustrated by the examples of the present application illustrate the strong potential of the application of Bacillus strains for the reduction of solanine formation and greening. It is to be noted that the final level of solanine in the tuber flesh is driven by variety. Recent new registered varieties tend to have lower solanine levels compared to older varieties. These new variety releases reflect the current breeding programs to lower solanine content.
Accordingly, the present invention also relates to the use of a Bacillus strain for prolonging storage stability of potatoes, wherein such prolongation is caused by delayed greening and/or formation of solanine.
An application prior to planting may be done at any time post-harvest of potato tubers, cut potato pieces, apical cuttings or seeds to be planted. Accordingly, an application could take place directly after harvest as the earliest point in time, during storage or directly prior to planting the tubers into the soil. To save work, it is also possible to apply at the time of planting, that is at the same time as the tubers, tuber pieces, potato transplants, apical cuttings or seeds are planted into the soil. Apical cuttings are rooted transplants produced from tissue culture plants (see VanderZaag et al. ,2021 in Solanum tuberosum - A Promising Crop for Starvation Problem.; ISBN 978-1-83969-167-6; and Buckseth et al., 2022; Frontiers in Agronomy 4:956667. doi: 10.3389/fagro.2022.956667) and can also advantageously be used in connection with the present invention.
Application may be made by a seed or tuber treatment and/or as a soil treatment and/or treatment of artificial soil substrates (e.g., rockwool, perlite, glass, and coconut fiber) or treatment of circulation water in hydroponic cultures. The Bacillus strain, in particular Bacillus subtilis sp. strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010, can be applied to a potato tuber or potato seed, and/or a locus on which the plant grows, such as soil. The Bacillus strain, in particular Bacillus subtilis sp. strain QST713 Bacillus amyloliquefaciens strain ATB-BAS-010 can be applied by any known means, such as by spraying a solution on the soil, soil surface drench, shanked-in, injected, foamed in, dipped in, applied in-furrow, as a band application along the seeding/planting line, sprayed, coated onto the tubers and/or applied by mixture with irrigation water. Application is preferably effected by spraying in furrow or dipping, or spraying or foaming the tubers or seeds.
For spraying, tubers are sprayed prior to planting into the soil with an effective amount of a Bacillus strain, in particular Bacillus subtilis sp. strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010. When sprayed, at least 80%, preferably at least 90%, at least 95% or even more of the surface of the tuber are moistened during the treatment. The same applies for botanical seeds. In practice, tubers are sprayed on the planter immediately prior to planting or sprayed in furrow. In-furrow application is effected through a nozzle system on the potato planter. The Bacillus strain, in particular the B. subtilis QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 product is suspended in water and the solution is sprayed on the soil that is used to cover the tubers (the ridge of the row). For treatment on the planter, the tubers are sprayed with a respective solution through nozzles on the planters that are directed to the tubers. Alternatively, tubers are treated during storage or after storage and before planting. Here, the spinning disc technology enables for low volume treatment and is effected while unloading storage and/or preparing the tubers for planting. Here, 0,5 to 1.5 l ofsolution is applied per ton of tubers. Alternatively, the treatment can be effected by using standard nozzles operating system with volumes ranging from 5 - 10 liter of solution per ton of tubers.
For application by dipping, at least 50%, preferably at least 60%, more preferably at least 80% or at least 90% of the surface of the tuber or seed is wetted. The dipping time is usually between 10 seconds and 5 minutes, preferably between 30 seconds and 3 minutes but may vary and be higher or lower depending on the variety and environmental conditions. One single application is normally sufficient and preferred. However, under certain circumstances a second or third application may be advisable or necessary in order to obtain the desired result.
The microorganisms and particular strains described herein, unless specifically noted otherwise, are all separated from nature and grown under artificial conditions such as in shake flask cultures or through scaled-up manufacturing processes, such as in bioreactors to maximize bioactive metabolite production, for example. Growth under such conditions leads to strain "domestication." Generally, such a "domesticated" strain differs from its counterparts found in nature in that it is cultured as a homogenous population that is not subject to the selection pressures found in the natural environment but rather to artificial selection pressures.
As used herein, the verb "comprise" as is used in this description and in the claims and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
In some embodiments, the compositions comprising a Bacillus strain, in particular QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or a mutant thereof, are liquid formulations. Nonlimiting examples of liquid formulations include suspension concentrations and oil dispersions. In other embodiments, the compositions are solid formulations. Non-limiting examples of solid formulations include freeze-dried powders and spray-dried powders.
Said Bacillus strain, in particular Bacillus subtilis sp. Strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or mutant thereof is generally applied at a rate of between 3,00E+05 colony forming units (cfu)/ha and 5,00E+10 cfu/ha when treated in furrow or between 5,00E+08 cfu/ton tubers and l,00E+l 1 cfu/ton tubers when dipped or sprayed or l,00E+5 - l,00E+8 cfu / 1000 grams of botanical seeds Application rates per row meter depend on row distance and accordingly the number of rows per hectare as the application rate per hectare remains the same regardless of row distance.
In particular, application rates also depend on the product comprising Bacillus strain, in particular B. subtilis QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or a mutant thereof.
For full field treatment, the Bacillus strain, in particular Bacillus subtilis sp. Strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or mutant thereof is generally applied at a rate of between 3,00E+09 colony forming units (cfu)/ha and 5,00E+9 cfu/ha when treated as full field application prior to planting. For in furrow treatment, rates for the product Serenade ASO comprising B. sub tilts QST713 range between 3,00E+07 cfu/ha and l,00E+10 cfu/ha, preferably between 3,00E+08 cfu/ha and 8,00E+09 cfu/ha, more preferably between 3,00E+09 cfu/ha and 5,00E+09. For a row distance of 66 cm, this most preferred range corresponds to between l,98E+05 and 3,30E+05 cfu/m row, for 75 cm to between 2,25E+05 and 3,75E+05 cfu/m row and for 90 cm to between 2,70E+05 and 4,50E+05 cfu/m row.
Alternatively, for in furrow treatment, rates for the product Serenade ASO comprising B. subtilis QST713 range between 3,00E+10 cfu/ha and l,00E+13 cfu/ha, preferably between 3,00E+l 1 cfu/ha and 8,00E+12 cfu/ha, more preferably between 3,00E+12 cfu/ha and 5,00E+12. For a row distance of 66 cm, this most preferred range corresponds to between l,98E+08 and 3,30E+08 cfu/m row, for 75 cm to between 2,25E+08 and 3,75E+08 cfu/m row and for 90 cm to between 2,70E+08 and 4,50E+08 cfu/m row.
For another formulation of QST713 (HiCFU) rates range between l,50E+09 cfu/ha and 3,00E+l 1 cfu/ha, preferably between l,00E+10 cfu/ha and 5,00E+10 cfu/ha, more preferably between l,50E+10 cfu/ha and 3,00E+10 when treated in furrow. For a row distance of 66 cm, this most preferred range corresponds to between 9,90E+05 and l,98E+06 cfu/m row, for 75 cm to between l,13E+06 and 2,25E+06 cfu/m row and for 90 cm to between l,35E+06 and 2,70E+06 cfu/m row.
Alternatively, for the HiCFU formulation, rates range between l,50E+12 cfu/ha and 3,00E+14 cfu/ha, preferably between l,00E+13 cfu/ha and 5,00E+13 cfu/ha, more preferably between l,50E+13 cfu/ha and 3,00E+13 when treated in furrow. For a row distance of 66 cm, this most preferred range corresponds to between 9,90E+08 and l,98E+09 cfu/m row, for 75 cm to between l,13E+09 and 2,25E+09 cfu/m row and for 90 cm to between l,35E+09 and 2,70E+09 cfu/m row.
For tuber treatment, rates for the product Serenade ASO range between 5,00E+07 cfu/ton tubers and l,00E+10 cfu/ton tubers, preferably between l,00E+08 cfu/ton tubers and 5,00E+09 cfu/ton tubers, more preferably between 5,00E+08 cfu/ton tubers and l,00E+09 cfu/ton tubers. For another formulation of QST713 (HiCFU) rates for tuber application range between 7,00E+08 cfu/ton tubers and l,50E+l 1 cfu/ton tubers, preferably between l,00E+09 cfu/ton tubers and l,00E+l 1 cfu/ton tubers, more preferably between 7,50E+09 cfu/ton tubers and l,50E+10 cfu/ton tubers.
Alternatively, for tuber treatment, rates for the product Serenade ASO range between 5,00E+10 cfu/ton tubers and l,00E+13 cfu/ton tubers, preferably between l,00E+ll cfu/ton tubers and 5,00E+12 cfu/ton tubers, more preferably between 5,00E+l 1 cfu/ton tubers and l,00E+12 cfu/ton tubers. For another formulation of QST713 (HiCFU) rates for tuber application range between 7,00E+ll cfu/ton tubers and l,50E+14 cfu/ton tubers, preferably between l,00E+12 cfu/ton tubers and l,00E+14 cfu/ton tubers, more preferably between 7,50E+12 cfu/ton tubers and l,50E+13 cfu/ton tubers. For in furrow treatment, rates for the product Rhizofert comprising Bacillus amyloliquefaciens, ATB - BAS-010, range between 3,00E+10 cfu/ha and l,00E+13 cfu/ha, preferably between 3,00E+l 1 cfu/ha and 8,00E+12 cfu/ha, more preferably between 3,00E+12 cfu/ha and 5,00E+12. For a row distance of 66 cm, this most preferred range corresponds to between l,98E+08 and 3,30E+08 cfu/m row, for 75 cm to between 2,25E+08 and 3,75E+08 cfu/m row and for 90 cm to between 2,70E+08 and 4,50E+08 cfu/m row.
For tuber treatment, rates for the product Rhizofert comprising Bacillus amyloliquefaciens, ATB -BAS- 010, range between 5,00E+10 cfu/ton tubers and l,00E+13 cfu/ton tubers, preferably between l,00E+l l cfu/ton tubers and 5,00E+12 cfu/ton tubers, more preferably between 5,00E+l l cfu/ton tubers and l,00E+12 cfu/ton tubers.The Bacillus strain, in particular B. subtilis strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or a mutant thereof, may also be applied to soil and/or plants in plug trays or to seedlings prior to transplanting to a different plant locus. When applied to the soil in contact with the plant roots, to the base of the plant, or to the soil within a specific distance around the base of the plant, including as a soil drench treatment, the strain may be applied as a single application or as multiple applications. The strain may be applied at the rates set forth above for drench treatments or a rate of about 1 x 105 to about 1 x 108 cfu per gram of soil, 1 x 105 to about 1 x 107 cfu per gram of soil, 1 x 105 to about IxlO6 cfu per gram of soil, 7 x 105 to about 1 x 10 7 cfu per gram of soil, 1 x 106 to about 5 x 106 cfu per gram of soil, or 1 x 105 to about 3 x 106 cfu per gram of soil. In one embodiment, the strain is applied as a single application at a rate of about 7 x 105 to about 1 x 107 cfu per gram of soil. In another embodiment, the strain is applied as a single application at a rate of about 1 x 106 to about 5 x 106 cfu per gram of soil. In other embodiments, the strain is applied as multiple applications at a rate of 10 about 1 x 105 to about 3 x 106 cfu per gram of soil.
The strains suitable for the present invention can be applied to botanical potato seeds using conventional treating techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed treaters, and drum coaters. Other methods, such as spouted beds may also be useful. The seeds may be pre-sized before coating. After coating, the seeds are typically dried and then transferred to a sizing machine for sizing. Such sizing and treating procedures are known in the art.
Although it is believed that the present method can be applied to a botanical potato seed in any physiological state, it is preferred that the seed be in a sufficiently durable state that it incurs no damage during the treatment process. Typically, the seed would be a seed that had been harvested from the field; removed from the plant; and separated from any cob, stalk, outer husk, and surrounding pulp or other nonseed plant material. The seed would preferably also be biologically stable to the extent that the treatment would cause no biological damage to the seed. It is believed that the treatment can be applied to the seed at any time between harvest of the seed and sowing of the seed or during the sowing process (seed directed applications). The seed may also be primed according to techniques understood by those skilled in the art either before or after the treatment. Even distribution of the active ingredients and adherence thereof to the seeds is desired during propagation material treatment. Treatment could vary from a thin fdm (dressing) of the formulation containing the strains suitable in the present invention on a plant propagation material, such as a seed, where the original size and/or shape are recognizable to an intermediary state (such as a coating) and then to a thicker fdm (such as pelleting) with many layers of different materials (such as carriers, for example, clays; different formulations, such as of other active ingredients; polymers; and colorants) where the original shape and/or size of the seed is no longer recognizable.
In some embodiments, the seed treatment occurs to an unsown seed. The term "unsown seed" is meant to include seed at any period between the harvest of the seed and the sowing of the seed in the ground for the purpose of germination and growth of the plant. Treatment to an unsown seed is not meant to include those practices in which the active ingredient is applied to the soil but would include any application practice that would target the seed during the planting process.
In some embodiments, treatment occurs before sowing of the seed so that the sown seed has been pretreated with the strains according to the present invention. In particular, seed coating or seed pelleting are preferred in the treatment with the strains described herein. As a result of the treatment, the strains suitable in the present invention are adhered onto the surface of the seed and therefore available for pest and/or disease control and for acting in reducing greening and solanine formation in the tubers.
The treated seeds can be stored, handled, sown and tilled in the same manner as any other active ingredient treated seed.
Potato varieties that can be used in the present invention are not limited. European potoato cultivars that can be used are listed, e.g., in the European Cultivated Potato Database (ECPD available under https://en.wikipedia.org/wiki/European_Cultivated_Potato_Database) or the EU Plant Variety Database available under https://ec.europa.eu/food/plant/plant_propagation_material/plant_variety_catalogues_databases/search/p ublic/index.cfm?event=SearchVariety&ctl_type=A&species_id=262&variety_name=&listed_in=0&sho w_current=on&show_deleted=. North American cultivars can be found in the Northe America Potato Variety Inventors (https://potatoassociation.org/publications-2/north-american-potato-variety- inventory/north-american-potato-variety-inventory-c-f/). Preferably, said potato variety is selected from the group consisting of table, processing and starch/protein potatoes. Exemplary variants include for table potatoes Allians, Alouette, Annabelle, Arizona, Amora, Aster, Belana, Bintje, Cammeo, Carolus, Connect, Desiree, Fabula, Gala, Granola, Irish Cobbler, Jazzy, Lady Anna, Manitou, Melody, Mila, Muse, Nicola, Panamera, Ratte, Sagita, Shangi, Spunta, Unica, Twister and Vitabella. Processing potato variants include Agate, Agria, Alpha, Atlantic, Asterix, Challenger, Fiona, Favourita, Fontane, Hansa, Hermes, Innovator, Ivory Russet, Jelly, Markies, Maris Piper, Mondial, Kennebec, King Edward, Lady Claire, Norland, Royal, Russet Burbank, Russet Norkotah, Ranger Russet, Shepody, Snowdon, Umatilla Russet, Vivaldi and Yukon Gold. Starch potatoes include the variants Altus, Avamond, Avama, Avatar, Avito, Axion, BMC, BMC, Festien, Kardal, Kuras, Saprodi, Seresta and Supporter.
In course of the invention, it was surprisingly found that Bacillus subtilis sp. strain QST713 that, inter alia, is known to have fungicidal action significantly delays greening and reduces the extent of greening and the content of solanine when applied pre planting. This results in a prolonged storage stability of potato tubers. Most notably, it was observed that the effect is passed on from a tuber treated and planted to daughter tubers harvested from the plant arising from said tuber. Without wishing to be bound by any scientific theory, it is believed that Bacillus subtilis sp. strain QST713 or mutants thereof colonize the tubers and later on the relevant plant parts and are thereby passed over to offspring of the original treated tuber.
Deposit Information
A sample of a Bacillus subtilis strain suitable in the present invention has been deposited with the Agricultural Research Service Culture Collection located at the National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604, U.S.A., under the Budapest Treaty on March 7, 1997, and has been assigned Accession Number NRRL B-21661.
Samples of QST30002 (aka AQ30002) and QST30004 (aka AQ30004) have been deposited with the Agricultural Research Service Culture Collection under the Budapest Treaty on October 5, 2010 and December 6, 2010, respectively. QST30002 has been assigned Accession Number NRRL B-50421, and QST30004 has been assigned the following Accession Number NRRL B-50455.
The Bacillus subtilis strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. § 122. The deposits represent a substantially pure culture of the deposited Bacillus subtilis strain. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. The following examples are given for purely illustrative and non-limiting purposes of the present invention.
Example 1: MALDI mass spectrometry imaging analysis for visualization of reducing in greening and/or formation of glycoalkaloids
The effect of storage time on glycoalkaloids for potato storage has a high impact on food safety. MALDI mass spectrometry imaging (MALDI MSI) allows an in-situ detection and imaging method for glycoalkaloids, which can visualize the spatial distribution and relative content changes of glycoalkaloids in potato tubers.
Tubers for cryosectioning were embedded in carboxymethylcellulose sodium salt (4 - 6% in water) and frozen at -20 °C for at least 12 hours. Samples were equilibrated in a cryostat CryoStar NX70 (Thermo Scientific GmbH, Bremen, Germany) for approximately 30 min at the optimal cutting temperature (-25 - -15 °C). Histological slices (20 - 35 pm) of tubers were mounted either on microscope slides (18x18x1 mm, Paul Marienfeld GmbH & Co. KG, Lauda-Kbnigshofen, Germany) directly or using thin doublesided adhesive tape (3M, Saint Paul, USA). The samples were dried in a desiccator at room temperature for a minimum of 30 min before matrix deposition. MALDI matrices were sprayed on the sample using a MALDI sprayer SunCollect (SunChrom Wissenschaftliche Gerate GmbH, Friedrichsdorf, Germany) and matrix flow rate, sample speed and spray intervals, optimized for each matrix and tissue. Before and after matrix deposition, the sample surfaces were checked with a digital microscope VHX-6000 (Keyence Deutschland GmbH, Neu-Isenburg, Germany). The matrix-coated target was stored in a vacuum desiccator at room temperature in the dark until analysis. According to the actual storage time of potato tubers, we chose 0, 7, 14, 21 and 28 days at room temperature for the experiment. Glycoalkaloids began to biosynthesize and increase and showed obvious distribution characteristic in potato tuber tissues. As visible in the mass spectrometric images, the relative content of glycoalkaloids in untreated potato tubers increases significantly. Glycoalkaloids were detected in the skin, periderm and medulla with high signal intensities for the untreated tubers. On the other hand, tubers treated with Bacillus subtilis sp. Strain QST713 showed less accumulation in the periderm and medulla. The spatial distribution and content of a-solanine ([M+H]+ at m/z 868.50529) and a-chaconine ([M+H]+ at m/z 852.51038) was investigated in potato tubers under different storage times. While the tubers treated with Bacillus subtilis sp. Strain QST71 refers to a reduction in greening and formation of a-solanine and a-chaconine of at least 5% post-harvest, the reducing in the whole potato tuber after 7 days is at least 45%. During the storage period the effect on reducing in greening and content of a-solanine and a-chaconine is at least 70% after 14 days and at least 80% up to 28 days. Table 1. Relative reducing in greening and formation of a-solanine and a-chaconine in potato tubers of different varieties (Saprodi, Fontana and Avama) treated with Bacillus subtilis sp. Strain QST713 after storage under UV light.
Figure imgf000015_0001
In summary, the spatial distribution of the glycoalkaloids a-solanine and a-chaconine in skin, periderm and medulla of potato tubers showed higher contents and higher accumulation in untreated tubers than in treated with Bacillus subtilis sp. Strain QST713 after 21 days storage times.

Claims

Claims
1. Use of a Bacillus strain to delay greening and/or formation of solanine in and/or for prolonging storage stability of potatoes caused by delayed greening and/or formation of solanine.
2. Use according to claim 1, wherein said Bacillus strain belongs to a species selected from the group consisting of . Bacillus subtilis var. amyloliquefaciens, Bacillus amyloliquefaciens and Bacillus sub tills
3. Use of claim 1, wherein said Bacillus strain is selected from the group consisting of Bacillus subtilis var. amyloliquefaciens strain FZB24, Bacillus amyloliquefaciens strain FZB42, Bacillus amyloliquefaciens strain D747, Bacillus amyloliquefaciens strain ATB-BAS-010, Bacillus subtilis strain Y1336, Bacillus subtilis strain MBI 600, Bacillus subtilis strain QST713 (Accession No. NRRU B-21661), Bacillus subtilis AQ30002 (Accession No. NRRU B-50421), Bacillus subtilis AQ30004 (Accession No. NRRU B-50455), Bacillus pumilus QST2808 (Accession No. NRRU B-30087), mutants thereof having all the identifying characteristics of the respective strain, and combinations thereof.
4. Use according to claim 1 or 2, wherein said Bacillus strain is Bacillus subtilis strain QST713, Bacillus amyloliquefaciens strain ATB-BAS-010 or a mutant thereof having all the identifying characteristics of the strain.
5. Method of delaying and/or reducing greening and/or formation of solanine in and/or for prolonging storage stability of potatoes comprising applying prior to or at the time of planting an effective amount of a Bacillus strain to potato tubers, tuber pieces, potato transplants, apical cuttings or botanical potato seeds, planting said tubers, tuber pieces, potato transplants, apical cuttings or seeds into the soil to let plants grow and obtaining potato tubers from said plants.
6. Method according to claim 4, wherein said Bacillus strain is selected from the group consisting of Bacillus subtilis var. amyloliquefaciens strain FZB24, Bacillus amyloliquefaciens strain FZB42, Bacillus amyloliquefaciens strain D747, Bacillus amyloliquefaciens strain ATB-BAS- 010, Bacillus subtilis strain Y1336, Bacillus subtilis strain MBI 600, Bacillus subtilis strain QST713 (Accession No. NRRU B-21661), Bacillus subtilis AQ30002 (Accession No. NRRU B- 50421), Bacillus subtilis AQ30004 (Accession No. NRRU B-50455), Bacillus pumilus QST2808 (Accession No. NRRU B-30087), mutants thereof having all the identifying characteristics of the respective strain, and combinations thereof.
7. Method according to claim 4 or 5, wherein said Bacillus strain is Bacillus subtilis strain QST713, Bacillus amyloliquefaciens strain ATB-BAS-010 or a mutant thereof having all the identifying characteristics of the strain.
8. The method according to any one of claims 4 to 6, wherein said Bacillus subtilis strain is applied as a soil surface drench, shanked-in, injected, foamed in, dipped in, applied in-furrow, as a band application along the seeding/planting line, sprayed, coated onto said tubers or tuber pieces and/or applied by mixture with irrigation water.
9. Method according to any one of claims 4 to 7, wherein said Bacillus strain is applied at a rate of between 3,00E+05 cfu/ha and 5,00E+10 cfu/ha when treated in furrow or between 5,00E+08 cfu/ton tubers and l,00E+l 1 cfu/ton tubers when dipped or sprayed or between l,00E+5 - l,00E +8 cfu / 1000 grams of botanical seeds
10. Use of any one of claims 1 to 3 or method of any one of claims 4 to 8, wherein the potatoes are selected from the group consisting of table potatoes, processing potatoes and starch/protein potatoes.
11. Use or method according to claim 9, wherein the potato variety is selected from the groups consisting of Allians, Alouette, Annabelle, Arizona, Amora, Aster, Belana, Bintje, Cammeo, Carolus, Connect, Desiree, Fabula, Gala, Granola, Irish Cobbler, Jazzy, Lady Anna, Manitou, Melody, Mila, Muse, Nicola, Panamera, Ratte, Sagitta, Shangi, Spunta, Unica, Twister, Vitabella, Agate, Agria, Alpha, Atlantic, Asterix, Challenger, Fiona, Favourita, Fontane, Hansa, Hermes, Innovator, Ivory Russet, Jelly, Markies, Maris Piper, Mondial, Kennebec, King Edward, Lady Claire, Norland, Royal, Russet Burbank, Russet Norkotah, Ranger Russet, Shepody, Snowdon, Umatilla Russet, Vivaldi, Yukon Gold, Altus, Avamond, Avama, Avatar, Avito, Axion, BMC, BMC, Festien, Kardal, Kuras, Saprodi, Seresta and Supporter.
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