Use for delaying greening and / or solanine formation of potatoes

Abstract

Use for delaying greening and / or solanine formation of potatoes. The invention relates to a method for delaying and / or reducing the greening and / or solanine formation of potatoes and / or for extending the storage stability of potatoes, the method comprises applying an effective amount of a bacillus strain to a potato tuber, a tuber cut, a potato transplanted seedling, an apical cottage or a botanical potato seed before or during planting, planting the tuber or seed into soil to grow the plant, and obtaining a potato tuber from the plant.

Description

[0001] During potato storage, tuber greening is often observed when certain environmental factors such as temperature and light are not set correctly. This is not just an aesthetic issue that is not well-received by consumers. However, greening is also associated with the presence of relatively large amounts of colorless glycoalkaloids, such as mainly α-solanine and α-chaconine. These glycoalkaloids impair flavor by causing bitterness and can cause consumer poisoning at concentrations above 200 mg per kg 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). Therefore, there is an urgent need to reduce or delay the greening process and glycoalkaloid content of tubers, regardless of storage conditions.

[0002] The present invention solves this technical problem at least in part.

[0003] Accordingly, the present invention relates to a method for delaying and / or reducing potato greening and / or solanine formation and / or for extending the storage stability of potatoes, the method comprising applying an effective amount of a Bacillus strain to potato tubers, tuber pieces, potato transplant seedlings, apical cuttings or botanical potato seeds before or at the time of planting, planting the tubers, tuber pieces, potato transplant seedlings, apical cuttings or botanical seeds into soil for plant growth, and obtaining potato tubers from the plants. The present invention also relates to the use of Bacillus subtilis strain QST713 for delaying and / or reducing potato greening and / or solanine formation and / or for extending the storage stability of potatoes.

[0004] As used herein, the genus Bacillus refers to a genus of Gram-positive, rod-shaped bacteria that are members of the phylum Firmicutes. Bacteria of the genus Bacillus can be characterized and identified based on the nucleotide sequence of their 16S rRNA or a fragment thereof (e.g., a fragment of about 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, or 1500 nt of the 16S rRNA or rDNA nucleotide sequence). The Bacillus strains of the present invention can be any one of the following species: Bacillus acidiceler, Bacillus acidicola, Bacillus acidiproducens, B. aeolius, Bacillus aerius, Bacillus aerophilus, Bacillus agaradhaerens, Bacillus aidingensis, Bacillus akibai, Bacillus alcalophilus, Bacillus algicola, Bacillus alkalinitrilicus, Bacillus alkalisediminis, Bacillus alkalitelluris, Bacillus altitdinis, Bacillus alveayuensis, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus aquimaris, Bacillus arsenicus, Bacillus aryabhattai, Bacillus asahii, Bacillus atrophaeus, Bacillus aurantiacus, Bacillus azotoformans, Bacillus badius, Bacillus barbaricus, Bacillus bataviensis, Bacillus beijingensis, Bacillus benzoevorans, B. beveridgei, Bacillus bogoriensis, Bacillus boroniphilus, Bacillus butanolivorans, Bacillus canaveralius, Bacillus carboniphilus, Bacillus cecembensis, Bacillus cellulolyticus, B.Cellulosilyiicus, Bacillus cereus, Bacillus chagannorensis, Bacillus chungangensis, Bacillus cibi, Bacillus circulans, Bacillus clarkii, Bacillus clausii, Bacillus coagulans, B. coahuilensis, Bacillus cohnii, Bacillus decisifrondis, Bacillus decolorationis, Bacillus drentensis, Bacillus farraginis, Bacillus faslidiosus, Bacillus firmus, Bacillus flexus, Bacillus foraminis, Bacillus fordii, Bacillus fortis, B. fumarioli, Bacillus funiculus, Bacillus galactosidilyticus, B. galliciensis, Bacillus gelatini, Bacillus gibsonii, Bacillus ginsengi, Bacillus ginsengihumi, Bacillus graminis, Bacillus halmapalus, B. halochares, Bacillus halodurans, Bacillus hemicellulosilyticus, B. herbertsteinensis, Bacillus horikoshi, Bacillus horneckiae, Bacillus horti, Bacillus humi, Bacillus hwajinpoensis, Bacillus idriensis, Bacillus indicus, Bacillus infantis, Bacillus infernus, Bacillus isabeliae, B. isronensis, Bacillus jeotgali, Bacillus koreensis, Bacillus korlensis, Bacillus mucilaginosus,B. kribbensis, B. krulchiae, B. lehensis, B. lentus, B. licheniformis, B. litoralis, B. locisalis, B. luciferensis, B. luteolus, B. macauensis, B. macyae, B. mannanilyticus, B. marisflavi, B. marmarensis, B. massiliensis, B. megaterium, B. methanolicus, B. methylotrophicus, B. mojavensis, B. muralis, B. murimartini, B. mycoides, B. nanhaiensis, 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. panaciterrae, B. patagoniensis, B. persepolensis, B. plakortidis, B. pocheonensis, B. polygoni, B. pseudoalcaliphilus, B. pseudofirmus, B. pseudomycoides, B. psychrosaccharolyticus, B. pumilus, B. qingdaonensisqingdaonensis), Bacillus aquarius, Bacillus agri, Bacillus arvi, Bacillus safensis, Bacillus salarius, Bacillus saliphilus, Bacillus schlegelii, Bacillus selenatarsenatis, Bacillus selenitireducens, Bacillus seohaeanensis, Bacillus shackletonii, Bacillus siamensis, Bacillus simplex, Bacillus siralis, Bacillus smithii, Bacillus soli, Bacillus solisalsi, Bacillus sonorensis, Bacillus sporothermodurans, Bacillus stratosphericus, Bacillus subterraneus, Bacillus subtilis, B. taeansis, Bacillus tequilensis, Bacillus thermantarcticus, Bacillus thermoamylovorans, Bacillus thermocloacae, Bacillus thermolactis, Bacillus thioparans, Bacillus thuringiensis, B. tripoxylicola, Bacillus tusciae, Bacillus vallismortis, Bacillus vedderi, Bacillus vietnamensis, Bacillus vireti, Bacillus wakoensis, Bacillus weihenstephanensis, Bacillus xiaoxiensis, and mixtures and blends thereof.

[0005] Suitable Bacillus strains are preferably strains of the following species: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus subtilis var. amyloliquefaciens, Bacillus pumilus, or combinations thereof. It should be understood that over time, certain Bacillus strains may be reclassified based on recent findings regarding genomic similarity and / or phylogeny. These reclassified strains are still included in the present invention. More preferably, the Bacillus strain is selected from: Bacillus subtilis var. amyloliquefaciens strain FZB24 (available as a fungicide or ECO obtained from Novozymes Biologicals Inc. (Salem, Virginia) or Syngenta Crop Protection, LLC (Greensboro, North Carolina) (EPA registration number 70127-5)), Bacillus amyloliquefaciens strain FZB42 (obtained as from Belchim), Bacillus amyloliquefaciens strain D747 (obtained as from New Zealand Etec Crop Solutions and also available as DOUBLE from Certis USA), Bacillus amyloliquefaciens strain ATB-BAS-010 (obtained as Rhizofert from Artechno, Gembloux), Bacillus subtilis Y1336 (obtained as WP is obtained from Bion-Tech in Taiwan, China, and is registered as a biocidal fungicide in Taiwan, China under registration numbers 4764, 5454, 5096, and 5277), Bacillus subtilis MBI 600 (which can be obtained as Integral Pro from BASF DE), Bacillus subtilis QST713 (accession number NRRL B-21661), Bacillus subtilis AQ30002 (accession number NRRL B-50421), Bacillus subtilis AQ30004 (accession number NRRL B-50455), Bacillus pumilus QST2808 (accession number NRRL B-30087), mutants thereof having all the identifying characteristics of their respective strains, and combinations thereof. Most preferably, the Bacillus strain is Bacillus subtilis strain QST713 (accession number NRRL B-21661) or Bacillus amyloliquefaciens strain ATB-BAS-010.

[0006] Bacillus subtilis QST713 has been deposited under accession number NRRL B-21661. Bacillus subtilis QST713, its mutants, its supernatant, and its lipopeptide metabolites, as well as methods for their use in controlling plant pathogens and insects, are fully described in U.S. Pat. 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 reference to QST713 in this specification refers to Bacillus subtilis QST713. Specific variants of Bacillus subtilis QST713 (e.g., Bacillus subtilis AQ30002 and AQ30004, which have been deposited under accession numbers NRRL B-50421 and NRRL B-50455) that are also suitable for the present invention are described in U.S. Patent Publication No. 2012 / 0231951.

[0007] When the corresponding US patent application No. 09 / 074,870 was filed in 1998, the strain was named Bacillus subtilis according to classical, physiological, biochemical and morphological methods. The taxonomy of the genus Bacillus has evolved since then, especially based on the progress of genetics and sequencing technologies, so the species naming is mainly based on DNA sequences rather than the methods used in 1998. After aligning the protein sequences of Bacillus amyloliquefaciens FZB42, Bacillus subtilis 168 and QST713, about 95% of the proteins found in Bacillus amyloliquefaciens FZB42 are 85% or more identical to the proteins found in QST713; while only 35% of the proteins of Bacillus subtilis 168 are 85% or more identical to the proteins in QST713. However, even with more reliance on genetics, there is still taxonomic ambiguity in the relevant scientific literature and regulatory documents, reflecting the development of the understanding of Bacillus taxonomy over the past years. For example, a pesticide product based on the Bacillus genus strain FZB24 (which has the same genetic relationship with QST713 as FZB42) is classified as Bacillus subtilis var. amyloliquefaciens in the documents of the US EPA. Due to the complexity of these names, according to the documents, this specific Bacillus genus species is named differently as Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus subtilis var. amyloliquefaciens. In addition, recent work aims to characterize the present strain QST713 as belonging to B. velenzis. Therefore, we retain the Bacillus subtilis naming for QST713 instead of changing it to Bacillus amyloliquefaciens or B. velenzis as currently expected based solely on sequence comparison and inferred taxonomy.

[0008] The product (US EPA registration number 69592-12) contains the patented strain of Bacillus subtilis (strain QST713) and many different lipopeptides that work together to disrupt disease pathogens and provide good antimicrobial activity. The product is used to protect plants (such as vegetable, potato, fruit, nut and vine crops) against diseases (such as fire blight, gray mold, sour rot, rust, sclerotinia, Rhizoctonia, powdery mildew, bacterial spot and white mold). Available The product is in liquid or dry formulation and can be applied as a foliar and / or soil treatment. Copies of the US EPA Master Labels of the product, including ASO, MAX and SERENADE Are publicly available through the USEPA / OPP Pesticide Product Label System (PPLS) of the National Pesticide Information Retrieval System (NPIRS).

[0009] The ASO (aqueous suspension - organic) contains 1.34% of dry QST713 as the active ingredient and 98.66% of other ingredients. The ASO is formulated to contain at least 1 x 10 9 cfu / g of QST713, and the maximum amount of QST713 has been determined to be 3.3 x 10 10 cfu / g. Alternative trade names for ASO include SERENADE SERENADE and GARDENDISEASE. For additional information, see the U.S. EPA Master Labels for ASO dated January 4, 2010, and the U.S. EPA Master Label for SERENADE which are each incorporated herein by reference in their entirety.

[0010] MAX contains 14.6% of dry QST713 as the active ingredient and 85.4% of other ingredients. MAX is formulated to contain at least 7.3 x 10 9 cfu / g of QST713, and the maximum amount of QST713 has been determined to be 7.9 x 10 10 cfu / g. For additional information, see the U.S. EPA Master Label for MAX which is incorporated herein by reference in its entirety.

[0011] OPTIMUM (or OPTI) contains 26.2% of dry QST713 as the active ingredient and 73.8% of other ingredients. OPTIMUM (or OPTI) is formulated to contain at least 1.31 x 10 10 cfu / g of QST713. For additional information, see the U.S. EPA Master Label for OPTIMUM (or OPTI) which is incorporated herein by reference in its entirety.

[0012] Compositions comprising 713 or mutants thereof can be obtained by culturing Bacillus subtilis QST713 or mutants thereof by methods known in the art, including using the media or other methods described in U.S. Patent No. 6,060,051. Conventional large-scale microbial culture methods include submerged fermentation, solid-state fermentation, or liquid surface culture. Near the end of fermentation, due to nutrient depletion, Bacillus subtilis cells begin to transition from the growth phase to the sporulation phase, such that the final products of fermentation are mainly spores, metabolites, and residual fermentation medium. Sporulation is part of the natural life cycle of Bacillus subtilis and is typically initiated by the cell's response to nutrient limitation. The fermentation is configured to obtain a high level of colony-forming units of Bacillus subtilis and to promote sporulation. The bacterial cells, spores, and metabolites in the medium obtained from fermentation can be used directly or concentrated by conventional industrial methods, including centrifugation, tangential flow filtration, depth filtration, and evaporation. The fermentation broth and the concentrated fermentation broth are both referred to herein as "fermentation products". The compositions used in the present invention include fermentation products. In some embodiments, the concentrated fermentation broth is washed, for example, by diafiltration, to remove residual fermentation broth and metabolites.

[0013] The fermentation broth or the concentrated fermentation broth can be dried using conventional drying processes or methods, with or without the addition of a carrier, such as spray drying, freeze drying, tray drying, fluidized bed drying, drum drying, or evaporation.

[0014] The FZB24 mutant, which has been assigned accession number 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 can be obtained from ABiTEP GMBH, Germany, as a plant strengthening product FZB42 is also described in European Patent Publication No. EP2179652 and Chen et al., “Comparative Analysis of the Complete Genome Sequence of the Plant Growth-Promoting Bacterium Bacillus amyloliquefaciens FZB42,” Nature Biotechnology, Vol. 25, No. 9 (September 2007). FZB42 mutants are described in International Publication No. WO 2012 / 130221, including Bacillus amyloliquefaciens ABI01, which was assigned the accession number DSM 10-1092 by DSMZ - German Collection of Microorganisms and Cell Cultures.

[0015] The term “mutant” refers to a genetic variant derived from Bacillus. In one embodiment, the mutant has all the identifying characteristics of Bacillus. In certain cases, the mutant has the ability to delay and / or reduce greening and / or solanine formation and / or extend storage stability. In another embodiment, the mutant is a genetic variant having a genomic sequence with a sequence identity greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98% or preferably greater than about 99% with a Bacillus isolate that promotes plant growth, and more preferably has the ability to reduce and / or delay potato greening and / or solanine formation at least as well as the parental QST713 strain. Mutants can be obtained by treating Bacillus cells having the above identifying characteristics with chemicals or radiation, or by selecting spontaneous mutants (such as mutants with phage resistance) from the cell population, or by other means known to those skilled in the art, and identifying the cells that still have the identifying characteristics. Targeted mutations can be introduced using CRISPR / Cas genome editing technology.

[0016] Delaying greening and / or solanine formation means that, compared to potatoes that are not treated with the present invention but are otherwise treated the same, one or both of them are delayed by at least 7 days, at least 15 days, at least 1 month, at least 2 months, at least 4 months or at least 8 months after harvest.

[0017] Reducing the greening of potatoes and / or the formation of glycoalkaloids such as solanine and / or chaconine means that, compared to potatoes that have not been treated according to the invention but are otherwise treated identically, the greening is reduced by at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 65%, most preferably at least 70% (both based on the fresh weight per potato) in the epidermis and / or immediately below the epidermis and / or in the vicinity of the epidermis (up to a distance of about 1.5 mm from the epidermis). Alternatively or additionally, reducing the formation of glycoalkaloids such as solanine and / or chaconine means that, compared to potatoes that have not been treated according to the invention but are otherwise treated identically, the content of solanine and / or chaconine in the whole potato tuber is reduced by at least 50%, or at least 60%, or at least 70%, preferably at least 75%, more preferably at least 80%, most preferably at least 85% (per potato fresh weight). The greening comparison can be made at any time point starting from 7 days after harvest. As is evident from the examples, the effect becomes more prominent with the extension of the storage time. Therefore, it is preferred to measure at least on the 14th day after harvest or even later.

[0018] The surface of the potato tuber is called the periderm and contains three different tissue types: the phellem (dead cells with corky cell walls), the phellogen, and the phelloderm. The internal tuber tissue is based on cortical cells. The synthesis of solanine occurs in the phellogen, the living cells of the phelloderm, and the cortical cells. The highest concentration of solanine is detected within 3 mm below the surface of the potato tuber. Several suitable methods for measuring the solanine content in potatoes are known, see for example EFSA Journal 2020, 18(8), 6222 (https: / / doi.org / 10.2903 / j.efsa.2020.6222). They include specific HPLC' / DAD / MS-based methods, described in Ieri et al. (2011; Food Chemistry 125, pp750 - 59).

[0019] Storage stability refers to the time during which potatoes can be stored without a significant reduction in quality. Such a significant reduction is generally considered to be indicated by the appearance of a certain degree of greening. Thus, greening is also associated with higher levels of, for example, solanine (see, e.g., Pavlista, 2001, Historical Materials from University of Nebraska-Lincoln Extension, page 88), and greening is also an indicator of solanine, which causes bitterness and can be toxic at high doses. Thus, the extended storage stability of the present invention mainly refers to the extension achieved by delaying potato greening, as, for example, achieved by the application of the present invention. In Europe, the maximum permitted level of solanine is currently set at 150 mg / kg of fresh potato weight, but this value may be reduced in the future. Thus, the storage stability of potatoes is defined as the time until the threshold of 150 mg of solanine / kg of fresh potato weight is reached. Preferably, the storage stability is the time until a threshold of 130 mg / kg, more preferably until 100 mg / kg. Thus, increasing the storage stability of potatoes means extending storage for at least 7 days, at least 15 days, at least 1 month, at least 2 months, at least 4 months or at least 8 months, wherein the content of greening or glycoalkaloids is reduced compared to potatoes not treated according to the present invention. The reduction in solanine content as shown in the examples of the present application illustrates the powerful effect of the application of Bacillus in reducing solanine formation and greening. It is noted that the final level of solanine in the tuber flesh is driven by the variety. Compared to older varieties, newly registered varieties tend to have lower solanine levels. The release of these new varieties reflects the current breeding programs for reducing solanine content.

[0020] Accordingly, the present invention also relates to the use of Bacillus for extending the storage stability of potatoes, wherein such extension is achieved by delaying greening and / or solanine formation.

[0021] Application can be carried out on potato tubers, potato cuttings, apical cuttings or seeds to be planted at any time after harvest. Thus, application can be carried out immediately at the earliest time point after harvest, during storage or immediately before planting the tubers into the soil. To save work, application can also be carried out at planting time, i.e., simultaneously with planting the tubers, tuber cuttings, potato transplants, apical cuttings or seeds into the soil.

[0022] The apical cuttings are root-bearing transplants propagated from tissue-cultured plants (see VanderZaag et al., 2021, 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 the cuttings can also be advantageously used in combination with the present invention.

[0023] Application can be carried out by seed or tuber treatment and / or soil treatment and / or treatment of artificial soil substrates (such as rock wool, perlite, glass, and coconut fiber) or treatment of the circulating water in hydroponic cultures. Bacillus strains, particularly Bacillus subtilis strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010, can be applied to potato tubers or potato seeds, and / or the site where the plants grow, such as the soil. The Bacillus strains, particularly Bacillus subtilis strain QST713, Bacillus amyloliquefaciens strain ATB-BAS-010, can be applied by any known method, such as by spraying a solution on the soil, surface drenching of the soil; shanked-in, injection, foaming, dipping, in-furrow application, band application along the sowing / planting line; spraying, coating onto the tubers, and / or by mixing with irrigation water. Application is preferably achieved by spraying or dipping in the furrow, or spraying or foaming the tubers or seeds.

[0024] Regarding spraying, tubers are sprayed with an effective amount of a Bacillus strain, in particular Bacillus subtilis strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010, before planting into the soil. When spraying is carried out, at least 80%, preferably at least 90%, at least 95% or even more of the tuber surface is wetted during the treatment. This also applies to plant seeds. In fact, the tubers are sprayed either on the seeder immediately before planting or in the furrow. The in-furrow application is carried out through the nozzle system on the potato seeder. The Bacillus strain, in particular the product of Bacillus subtilis QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010, is suspended in water and the solution is sprayed onto the soil (the ridge of the row) used to cover the tubers. Regarding the treatment on the seeder, the corresponding solution is sprayed onto the tubers through the nozzles on the seeder that point to the tubers. Alternatively, the tubers are treated during storage or after storage and before planting. Here, the rotary disc technology enables low-volume treatment and is carried out while unloading the stored and / or preparing the tubers for planting. Here, 0.5 to 1.5 L of the solution is applied per ton of tubers. Alternatively, the treatment can be achieved by using a standard nozzle operating system with a capacity of 5 - 10 liters of solution per ton of tubers.

[0025] Regarding application by immersion, at least 50%, preferably at least 60%, more preferably at least 80% or at least 90% of the tuber or seed surface is wetted. The immersion time is generally between 10 seconds and 5 minutes, preferably between 30 seconds and 3 minutes, but the immersion time can vary and increase or decrease according to the variety and environmental conditions.

[0026] Usually a single application is sufficient and preferably a single application. However, in some cases, a second or third application may be desirable or necessary to obtain the desired result.

[0027] Unless otherwise explicitly stated, the microorganisms and specific strains described herein are isolated from nature and grown under artificial conditions (such as in shake flask cultures) or by scale-up manufacturing methods (such as in bioreactors) to maximize the production of, for example, bioactive metabolites. Growth under such conditions results in "domestication" of the strain. Generally, such "domesticated" strains differ from their corresponding strains found in nature in that they are cultured as a homogeneous population that does not experience selection pressure based on the natural environment but rather artificial selection pressure.

[0028] As used herein, the verb "comprising" and variations thereof used in the specification and claims are used in their non-limiting sense, meaning including the items following the word, but not excluding items not specifically mentioned. In addition, the mention of an element by the indefinite article "a" or "an" does not exclude the possibility of more than one of the said elements being present, unless the context clearly requires that there be one and only one of the said elements. Thus, the indefinite article "a" generally means "at least one".

[0029] In some embodiments, the composition comprises a Bacillus strain, particularly Bacillus subtilis strain QST713 or Bacillus amyloliquefaciens strain ATB - BAS - 010 or a mutant thereof, and the composition is a liquid formulation. Non-limiting examples of liquid formulations include suspension concentrates and oil dispersions. In other embodiments, the composition is a solid formulation. Non-limiting examples of solid formulations include freeze-dried powders and spray-dried powders.

[0030] The Bacillus strain, particularly Bacillus subtilis strain QST713 or Bacillus amyloliquefaciens strain ATB - BAS - 010 or a mutant thereof, is generally applied at the following rates: when applied in-furrow, at a rate between 3.00E+05 colony forming units (cfu) / ha and 5.00E+10 cfu / ha, or when applied by dipping or spraying, at a rate between 5.00E+08 cfu / tonne of tubers and 1.00E+11 cfu / tonne of tubers, or at a rate of 1.00E+5 cfu / 1000 g of plant seeds to 1.00E+8 cfu / 1000 g of plant seeds. The application rate per row meter depends on the row spacing and accordingly the number of rows per hectare, as the application rate per hectare remains constant regardless of the variation in row spacing.

[0031] In particular, the application rate also depends on the product comprising the Bacillus strain, particularly Bacillus subtilis QST713 or Bacillus amyloliquefaciens strain ATB - BAS - 010 or a mutant thereof.

[0032] Regarding full-field treatment, when treated by full-field application before planting, the Bacillus strain, particularly Bacillus subtilis strain QST713 or Bacillus amyloliquefaciens strain ATB - BAS - 010 or a mutant thereof, is generally applied at a rate between 3.00E+09 colony forming units (cfu) / ha and 5.00E+09 cfu / ha.

[0033] For in-furrow treatment, the rate of the product Serenade ASO containing Bacillus subtilis QST713 ranges between 3.00E+07 cfu / ha and 1.00E+10 cfu / ha, preferably between 3.00E+08 cfu / ha and 8.00E+09 cfu / ha, and more preferably between 3.00E+09 cfu / ha and 5.00E+09 cfu / ha. For a row spacing of 66 cm, this most preferred range corresponds to between 1.98E+05 cfu / row-meter and 3.30E+05 cfu / row-meter; for 75 cm, it corresponds to between 2.25E+05 cfu / row-meter and 3.75E+05 cfu / row-meter; for 90 cm, it corresponds to between 2.70E+05 cfu / row-meter and 4.50E+05 cfu / row-meter.

[0034] Alternatively, for in-furrow treatment, the rate of the product Serenade ASO containing Bacillus subtilis QST713 ranges between 3.00E+10 cfu / ha and 1.00E+13 cfu / ha, preferably between 3.00E+11 cfu / ha and 8.00E+12 cfu / ha, and more preferably between 3.00E+12 cfu / ha and 5.00E+12 cfu / ha. For a row spacing of 66 cm, this most preferred range corresponds to between 1.98E+08 cfu / row-meter and 3.30E+08 cfu / row-meter; for 75 cm, it corresponds to between 2.25E+08 cfu / row-meter and 3.75E+08 cfu / row-meter; for 90 cm, it corresponds to between 2.70E+08 cfu / row-meter and 4.50E+08 cfu / row-meter.

[0035] For another formulation of QST713 (HiCFU), when used for in-furrow treatment, the rate ranges between 1.50E+09 cfu / ha and 3.00E+11 cfu / ha, preferably between 1.00E+10 cfu / ha and 5.00E+10 cfu / ha, and more preferably between 1.50E+10 cfu / ha and 3.00E+10 cfu / ha. For a row spacing of 66 cm, this most preferred range corresponds to between 9.90E+05 cfu / row-meter and 1.98E+06 cfu / row-meter; for 75 cm, it corresponds to between 1.13E+06 cfu / row-meter and 2.25E+06 cfu / row-meter; for 90 cm, it corresponds to between 1.35E+06 cfu / row-meter and 2.70E+06 cfu / row-meter.

[0036] Alternatively, for the HiCFU formulation, when applying in furrows, the rate ranges between 1.50E+12 cfu / ha and 3.00E+14 cfu / ha, preferably between 1.00E+13 cfu / ha and 5.00E+13 cfu / ha, more preferably between 1.50E+13 cfu / ha and 3.00E+13 cfu / ha. For a row spacing of 66 cm, this most preferred range corresponds to between 9.90E+08 cfu / row meter and 1.98E+09 cfu / row meter; for 75 cm, it corresponds to between 1.13E+09 cfu / row meter and 2.25E+09 cfu / row meter; for 90 cm, it corresponds to between 1.35E+09 cfu / row meter and 2.70E+09 cfu / row meter.

[0037] For tuber treatment, the rate of the product Serenade ASO ranges between 5.00E+07 cfu / tuber and 1.00E+10 cfu / tuber, preferably between 1.00E+08 cfu / tuber and 5.00E+09 cfu / tuber, more preferably between 5.00E+08 cfu / tuber and 1.00E+09 cfu / tuber. For another formulation of QST713 (HiCFU), the tuber application rate ranges between 7.00E+08 cfu / tuber and 1.50E+11 cfu / tuber, preferably between 1.00E+09 cfu / tuber and 1.00E+11 cfu / tuber, more preferably between 7.50E+09 cfu / tuber and 1.50E+10 cfu / tuber.

[0038] Alternatively, for tuber treatment, the rate of the product Serenade ASO ranges between 5.00E+10 cfu / tuber and 1.00E+13 cfu / tuber, preferably between 1.00E+11 cfu / tuber and 5.00E+12 cfu / tuber, more preferably between 5.00E+11 cfu / tuber and 1.00E+12 cfu / tuber. For another formulation of QST713 (HiCFU), the tuber application rate ranges between 7.00E+11 cfu / tuber and 1.50E+14 cfu / tuber, preferably between 1.00E+12 cfu / tuber and 1.00E+14 cfu / tuber, more preferably between 7.50E+12 cfu / tuber and 1.50E+13 cfu / tuber.

[0039] For in-furrow treatment, the rate of the product Rhizofert, which contains Bacillus amyloliquefaciens ATB-BAS-010, ranges between 3.00E+10 cfu / ha and 1.00E+13 cfu / ha, preferably between 3.00E+11 cfu / ha and 8.00E+12 cfu / ha, and more preferably between 3.00E+12 cfu / ha and 5.00E+12. For a row spacing of 66 cm, this most preferred range corresponds to between 1.98E+08 cfu / row-meter and 3.30E+08 cfu / row-meter; for 75 cm, it corresponds to between 2.25E+08 cfu / row-meter and 3.75E+08 cfu / row-meter; and for 90 cm, it corresponds to between 2.70E+08 cfu / row-meter and 4.50E+08 cfu / row-meter.

[0040] For tuber treatment, the rate of the product Rhizofert, which contains Bacillus amyloliquefaciens ATB-BAS-010, ranges between 5.00E+10 cfu / tuber and 1.00E+13 cfu / tuber, preferably between 1.00E+11 cfu / tuber and 5.00E+12 cfu / tuber, and more preferably between 5.00E+11 cfu / tuber and 1.00E+12 cfu / tuber. Bacillus strains, particularly Bacillus subtilis strain QST713 or Bacillus amyloliquefaciens strain ATB-BAS-010 or mutants thereof, can also be applied to the soil and / or plants in trays or to seedlings before transplanting to different planting sites. When applied to the soil in contact with the plant roots, the base of the plant, or the soil within a specific distance around the base of the plant, including in the form of a soil drench treatment, the strain can be applied once or multiple times. The strain can be applied at the rates used for drench treatment or at a rate of about 1×10 5 to about 1×10 8 cfu per gram of soil, about 1×10 5 to about 1×10 7 cfu per gram of soil, about 1×10 5 to about 1×10 6 cfu per gram of soil, about 7×10 5 to about 1×10 7 cfu per gram of soil, about 1×10 6 to about 5×10 6 cfu per gram of soil, or about 1×10 5 to about 3×10 6 cfu per gram of soil. In one embodiment, the strain is applied at a rate of about 7×10 5 to about 1×10 7The ratio of cfu is applied once. In another embodiment, the strain is applied at a ratio of about 1×10 6 to about 5×10 6 cfu per gram of soil once. In other embodiments, the strain is applied at a ratio of about 1×10 5 to about 3×10 6 cfu per gram of soil multiple times.

[0041] The strains suitable for the present invention can be applied to botanical potato seeds using conventional treatment techniques and machines, such as fluidized bed technology, roller milling, rotostatic seed processors, and drum coaters. Other methods, such as spouted beds, can also be used. The seeds can be pre-sized before coating. After coating, the seeds are usually dried and then transferred to a sizing machine for sizing. Such sizing and treatment procedures are known in the art.

[0042] Although the method is considered applicable to botanical potato seeds in any physiological state, it is preferred that the seeds are in a state that can tolerate sufficiently so as not to cause damage during the treatment process. Generally, the seeds can be seeds harvested from the field, seeds isolated from plants, seeds separated from any ear axis, stem, husk, and surrounding pulp or other non-seed plant materials. The seeds can also preferably be biologically stable to such an extent that the treatment does not cause biological damage to the seeds. It is believed that the seeds can be treated (for application to the seeds) at any time between seed harvest and seed sowing or during the sowing process. The seeds can also be germinated before or after treatment according to techniques known to those skilled in the art.

[0043] During the treatment of propagation materials, it is desired that the active ingredient is evenly distributed and adhered to the seeds. The treatment can vary from a film (seed coating) containing a preparation of the strain suitable for the present invention on plant propagation materials such as seeds (at this time, the original size and / or shape of the seeds are recognizable), to an intermediate state (such as coating), and then until a thicker film (such as granulation) with multiple layers of different materials (such as carriers, such as clay; different preparations, such as preparations of other active ingredients; polymers; and colorants) (at this time, the original shape and / or size of the seeds can no longer be recognized).

[0044] In some embodiments, the seed treatment occurs on unplanted seeds. The term "unplanted seeds" is intended to include seeds at any stage between seed harvest and sowing the seeds in the soil for plant germination and growth. The treatment of unplanted seeds is not intended to include those practices in which the active ingredient is applied to the soil, but will include any practices for application to the seeds during the planting process.

[0045] In some embodiments, the treatment occurs before sowing of the seeds, so that the sown seeds have been pretreated with the strains of the present invention. In particular, in the treatment with the strains described herein, seed coating or seed granulation is preferred. As a result of the treatment, the strains suitable for the present invention attach to the surface of the seeds and can thus be used for pest and / or disease control and for reducing greening and solanine formation in tubers.

[0046] The treated seeds can be stored, treated, sown and cultivated in the same manner as seeds treated with any other active ingredient.

[0047] The potato varieties that can be used in the present invention are not limited. European potato varieties that can be used are listed, for example, in the European Cultivated Potato Database (ECPD, which can be obtained at the following address: https: / / en.wikipedia.org / wiki / European_Cultivated_Potato_Database) or the EU Plant Variety Database (which can be obtained at the following address: https: / / ec.europa.eu / food / plant / plant_propagation_material / plant_variety_catalogues_databases / search / public / index.cfm?event=SearchVariety&ctl_type=A&species_id=262&variety_name=&listed_in=0&show_current=on&show_deleted=.). North American cultivated varieties can be seen in the North American Potato Variety Inventor (https: / / potatoassociation.org / publications-2 / north-american-potato-variety-inventory / north-american-potato-variety-inventory-c-f / ). Preferably, the potato variety is selected from table potatoes, processing potatoes, and starch / protein potatoes. Exemplary variants of table potatoes include Allians, Alouette, Annabelle, Arizona, Amora, Aster, Belana, Bintje, Cammeo, Carolus, Connect, Désirée, Fabula, Gala, Granola, Irish Cobbler, Jazzy, Lady Anna, Manitou, Melody, Mila, Muse, Nicola, Panamera, Ratte, Sagitta, 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 variants Altus, Avamond, Avarna, Avatar, Avito, Axion, BMC, BMC, Festien, Kardal, Kuras, Saprodi, Seresta and Supporter.

[0048] During the course of the present invention, it has surprisingly been found that the Bacillus subtilis strain QST713, which is known in particular for its fungicidal action, significantly delays greening and reduces the degree of greening and the content of solanine when applied before planting. This results in an extended storage stability of the potato tubers. Most strikingly, this effect has been observed to be transferred from the treated and planted tubers to the daughter tubers harvested from the plants produced by said tubers. Without wishing to be bound by any scientific theory, it is assumed that the Bacillus subtilis strain QST713 or a mutant thereof colonizes the tubers and subsequently colonizes the relevant plant parts, thereby being transferred to the offspring of the originally treated tubers.

[0049] Deposition information

[0050] A sample of the Bacillus subtilis strain suitable for the present invention was deposited on 7 March 1997 with the Agricultural Research Service Culture Collection Center in accordance with the Budapest Treaty. The said center is located at 1815 North University Street, Peoria, Illinois 61604, USA, at the National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, and has been assigned the accession number NRRL B-21661.

[0051] Samples of QST30002 (also known as AQ30002) and QST30004 (also known as AQ30004) were deposited at the Agricultural Research Service Culture Collection on October 5, 2010 and December 6, 2010, respectively, under the Budapest Treaty. QST30002 was assigned accession number NRRL B-50421 and QST30004 was assigned accession number NRRL B-50455.

[0052] The deposit conditions of the Bacillus subtilis strains ensure that cultures are available to persons determined to be eligible by the Commissioner of Patents and Trademarks under 37 C.F.R. § 1.14 and 35 U.S.C. § 122 during the pendency of this patent application. The deposits represent substantially pure cultures of the deposited Bacillus subtilis strains. The deposits are available subject to the requirements of foreign patent laws in the country where the corresponding application or its progeny applications to this application are filed. However, it should be understood that the availability of the deposits does not constitute a license to practice the invention without regard to any patent rights granted by government action.

[0053] The following examples are provided solely for purposes of illustration and are not limiting of the present invention.

[0054] Example 1: MALDI mass spectrometry imaging analysis to visualize the reduction of greening and / or glycoalkaloid formation

[0055] The effect of storage time on glycoalkaloids stored in potatoes has a great impact on food safety. Matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI MSI) allows in-situ detection and imaging of glycoalkaloids, which can visualize the spatial distribution and relative content changes of glycoalkaloids in potato tubers.

[0056] Tubers for cryosectioning were embedded in sodium carboxymethyl cellulose (4 - 6% in water) and frozen at -20 °C for at least 12 hours. Samples were equilibrated for approximately 30 minutes at the optimal sectioning temperature (-25 - -15 °C) in a CryoStar NX70 cryostat (Thermo Scientific GmbH, Bremen, Germany). Histological sections (20 - 35 μm) of the tubers were mounted directly on microscope slides (18 x 18 x 1 mm, Paul Marienfeld GmbH & Co. KG, Lauda- Germany) or using thin double-sided tape (3M, Saint Paul, USA). Prior to matrix deposition, the samples were dried in a desiccator at room temperature for at least 30 minutes. Using a MALDI sprayer SunCollect (SunChrom Wissenschaftliche GmbH, Friedrichsdorf, Germany) sprayed the MALDI matrix on the samples and optimized the matrix flow rate, sample speed, and spraying interval for each matrix and tissue. Before and after matrix deposition, the sample surface was examined using a digital microscope VHX-6000 (Keyence Deutschland GmbH, Neu-Isenburg, Germany). The matrix-coated targets were stored in a vacuum desiccator at room temperature in the dark until analysis. Depending on the actual storage time of the potato tubers, we chose to conduct the experiments at 0, 7, 14, 21, and 28 days at room temperature. Glycoalkaloids began to biosynthesize and increase in potato tuber tissue and exhibited distinct distribution characteristics. As visible in the mass spectrometry images, the relative content of glycoalkaloids in untreated potato tubers increased significantly. For untreated tubers, glycoalkaloids were detected at high signal intensities in the epidermis, periderm, and medulla. On the other hand, tubers treated with Bacillus subtilis strain QST713 showed less accumulation in the periderm and medulla. The spatial distribution and content of α-solanine ([M+H] + at m / z868.50529) and α-chaconine ([M+H] + at m / z 852.51038) in potato tubers at different storage times were studied. Tubers treated with Bacillus subtilis strain QST71 showed at least a 5% reduction in post-harvest greening and the formation of α-solanine and α-chaconine, and at least a 45% reduction throughout the potato tubers after 7 days. During storage, the effects of reducing greening and the content of α-solanine and α-chaconine were at least 70% after 14 days and at least 80% up to 28 days.

[0057] Table 1. Relative reduction in greening and the formation of α-solanine and α-chaconine after storage under ultraviolet light in potato tubers of different varieties (Saprodi, Fontana, and Avarna) treated with Bacillus subtilis strain QST71.

[0058] Number of storage days under ultraviolet light Saprodi Fontana Avarna 0 days 5％ 5％ 5 7 days 50％ 50％ 45％ 14 days 70％ 70％ 65％ 21 days 80％ 80％ 80％ 28 days 80％ 80％ 80％

[0059] In summary, the spatial distribution of the glycoalkaloids α-solanine and α-chaconine in the epidermis, periderm, and medulla of potato tubers showed that after 21 days of storage, the content and accumulation in untreated tubers were higher than those in tubers treated with Bacillus subtilis strain QST713.

Claims

1. Use of a Bacillus strain for delaying the greening and / or solanine formation of potatoes and / or for extending the storage stability of potatoes by delaying greening and / or solanine formation.

2. The use according to claim 1, wherein the Bacillus strain belongs to a species selected from the following genera: Bacillus subtilis var. amyloliquefaciens, Bacillus amyloliquefaciens, and Bacillus subtilis.

3. The use according to claim 1, wherein the Bacillus strain is selected from: Bacillus subtilis var. amyloliquefaciens strain FZB24, Bacillus amyloliquefaciens strain FZB42, Bacillus amyloliquefaciens strain D747, Bacillus amyloliquefaciens strain ATB - BAS - 010, Bacillus subtilis Y1336, Bacillus subtilis MBI 600, Bacillus subtilis QST713 (accession number NRRL B - 21661), Bacillus subtilis AQ30002 (accession number NRRL B - 50421), Bacillus subtilis AQ30004 (accession number NRRL B - 50455), Brevibacillus brevis QST2808 (accession number NRRL B - 30087), mutants thereof having all the identifying characteristics of the respective strains, and combinations thereof.

4. The use according to claim 1 or 2, wherein the Bacillus strain is Bacillus subtilis QST713, Bacillus amyloliquefaciens strain ATB - BAS - 010, or a mutant thereof having all the identifying characteristics of the strain.

5. A method for delaying and / or reducing the greening and / or solanine formation of potatoes and / or for extending the storage stability of potatoes, the method comprising applying an effective amount of a Bacillus strain to potato tubers, tuber cuttings, potato transplant seedlings, apical cuttings, or botanical potato seeds before or at the time of planting, planting the tubers, tuber cuttings, potato transplant seedlings, apical cuttings, or seeds into soil for plant growth, and obtaining potato tubers from the plants.

6. The method according to claim 4, wherein the Bacillus strain is selected from: Bacillus subtilis var. amyloliquefaciens strain FZB24, Bacillus amyloliquefaciens strain FZB42, Bacillus amyloliquefaciens strain D747, Bacillus amyloliquefaciens strain ATB - BAS - 010, Bacillus subtilis Y1336, Bacillus subtilis MBI 600, Bacillus subtilis QST713 (accession number NRRL B - 21661), Bacillus subtilis AQ30002 (accession number NRRL B - 50421), Bacillus subtilis AQ30004 (accession number NRRL B - 50455), Brevibacillus brevis QST2808 (accession number NRRL B - 30087), mutants thereof having all the identifying characteristics of the respective strains, and combinations thereof.

7. The method according to claim 4 or 5, wherein the Bacillus strain is Bacillus subtilis 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 the Bacillus subtilis strain is applied in the following manner: as a soil surface infusion; stabbing; injection; foaming; impregnation; in-furrow application; band application along the sowing / planting line; spraying; coating onto tubers and / or applying by mixing with irrigation water.

9. The method according to any one of claims 4 to 7, wherein the Bacillus strain is applied at a rate of 3.00E+05 cfu / ha to 5.00E+10 cfu / ha when performing in-furrow treatment, or at a rate of 5.00E+08 cfu / ton of tubers to 1.00E+11 cfu / ton of tubers when performing impregnation or spraying, or at a rate of 1.00E+5 cfu / 1000 g of plant seeds to 1.00E+8 cfu / 1000 g of plant seeds.

10. The use according to any one of claims 1 to 3 or the method according to any one of claims 4 to 8, wherein the potato is selected from table potatoes, processing potatoes, and starch / protein potatoes.

11. The use or method according to claim 9, wherein the potato varieties are selected from Allians, Alouette, Annabelle, Arizona, Amora, Aster, Belana, Bintje, Cammeo, Carolus, Connect, Désirée, 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, KingEdward, Lady Claire, Norland, Royal, Russet Burbank, Russet Norkotah, RangerRusset, Shepody, Snowdon, Umatilla Russet, Vivaldi, Yukon Gold, Altus, Avamond, Avarna, Avatar, Avito, Axion, BMC, BMC, Festien, Kardal, Kuras, Saprodi, Seresta, and Supporter.