Selection and use of cold-tolerant bacillus strains as biological phytostimulators (abitep) electricity symbols ks2

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The introduction of biological plant protection and organic fertilisers is an environmentally, friendly alternative to chemical pesticides and mineral fertilisers. “Plant-growth-promoting” rhizobacteria (PGPR) are used for the production of effective organic formulae. Members of the Bacillus type with the ability to form permanent endospores are of great importance since they have a natural high degree of stability. Spores are permanent forms of the bacterial cell, which can withstand extreme environmental influences such as heat and aridity, and can be stored for years) without a loss of activity.

Organic formulae of members of the Bacillus type are the most frequently used biological plant protection and phytostimulators (Borriss 2011). Aside from the known producers of the insecticides Bacillus thuringiensis and Bacillus kurstakii, these are strains of the Bacillus subtilis, family incl. B. amyloliquefaciens subsp. plantarum, B. subtilis, B. licheniformis and B. pumilus, which are most commonly used in this field. Examples of commercial products (organic fertilizers and/or plant protection) that contain B. amyloliquefaciens plantarum spores are Kodiak™ (Bayer Crop Science), Companion (Growth Products Ltd.), BioYield™ (Bayer Crop Science), INTEGRAL® (BASF), VAULT® (BASF), SERENADE Max® (Bayer Crop Science), CEASE® (BioWorks, Inc.), RhizoVital® (ABiTEP GmbH), FZB24® (ABiTEP GmbH), Double Nickel 55™ (Certis U.S.A.), Amylo-X® (Certis U.S.A.). Plant protection based on B. licheniformis are Green Releaf and EcoGuard (Novozyme Biologicals Inc.). The B. pumilus GB34 strain (Yield Shield, Bayer Crop Science) is an active ingredient of organic fungicides. Other EPO-registered B. pumilus organic fungicides are SONATA (Bayer Crop Science), and GHA 180 (Premier Horticulture). Members of B. mojavensis have also been described as photostimulatory and capable of endophytic growth (Bacon & Hinton 2002). Other bacilli which do not belong to the B. subtilis species complex also stimulate plant growth and plant health. B. firmus GB126 (BioNem AgroGreen, Israel now taken over by Bayer Crop Science) is used as an EPO-registered nematicide for the organic control of phytopathogenic nematodes. A similar product from B. firmus Bmj WG is currently being developed by Certis U.S.A. A phytostimulatory B. megaterium (López-Bucio et al. 2007) strain is the basis for the BioArc organic fungicide. Members of the Bacillus cereus species complex, e.g. B. cereus UW8 (Handelsman et al. 1990), B. thuringiensis and B. weihenstephanensis have also been described as plant growth promoting, endospore-forming rhizobacteria.

These bacteria used for producing the current organic products are known as “mesophile” bacteria and require growth temperatures of over 15° C. in order to develop their effect as phytostimulators. More than 85% of the Earth’s surface is covered by cold ecosystems, however, including Alpine mountain regions and areas that are oceanic and close to the poles (Feller & Gerday 2003). At temperature conditions below 15° C., which are typical for these regions, mesophile bacteria can have no positive effect on plant growth. An attractive alternative is the use of cold-tolerant microorganisms. Although their optimal growth conditions lie in the mesophile range, these microorganisms have developed mechanisms which give them the ability to continue to grow at lower temperatures (≤10°-0° C.) (Kumar et al. 2010). A note was made that the use of cold-tolerant and plant growth promoting bacteria as inoculants, in particular for agricultural production in tempered climate zones with low temperatures and a short growth season can be attractive (Mishra et al. 2012). However, the known examples for the use of cold-tolerant bacteria in plant production on gram-negative phytostimulatory bacteria such as Pseudomonas (Katiyar & Goel 2003), Burkholderia (Barka et al. 2006), Acinetobacter (Gulati et al. 2009), Azospirillum (Kaushik et al. 2002), Rhizobium (Prevost et al. 2003), Bradyrhizobium (Zhang et al. 2003), Pantoea (Selvakumar et al. 2008a), Serratia (Selvakumar et al. 2008b). Although in the past, several psychrophile or cold-tolerant Bacillus types, such as Bacillus globisporus (now Sporosarcina globisporus), Bacillus insolitus (now: Psychrobacillus insolitus), Paenibacillus macquariensis, Bacillus marinus (now: Marinibacillus marinus), Bacillus psychrophilus (now: Sporosarcina psychrophilus), Bacillus psychrosaccharolyticus, Bacillus psychrotolerans (now: Psychrobacillus psychrotolerans) and Bacillus psychrodurans (now: Psychrobacillus psychrodurans) have been described (El-Rahman et al. 2002, Krishnamurti et al. 2010), there is a lack of information regarding Bacillus strains with a phytostimulatory effect.

The goal of the invention is to develop an environmentally sensitive and sustainable means for stimulating plant growth, including at temperatures below 15° C. The means should in particular be used in climatically unfavourable regions such as mountain regions and areas relatively close to the poles of the northern and southern hemisphere.

The object is attained according to the claim; subclaims 2- 11 are preferred variants. The core of the invention is the use of cold-tolerant bacillus strains as biological phytostimulators. The scope of the invention further includes the combination of cold-tolerant bacillus strains with mesophile bacteria of the order Bacillales, preferably of type Bacillus and Paenibacillus, and the combination with humic acids. In the search for plant growth-promoting bacteria that show clear growth at temperatures below 15° C., it was found that endospore-forming bacilli that were isolated from cool climate regions such as mountain regions in the uplands of Tibet (People’s Republic of China) or the Alpine foothills (1400 m) are also able to grow at temperatures below 12° C. In accordance with their 16S rNA sequences, these cold-adapted bacteria were assigned to the types Bacillus simplex, Bacillus pumilus and to the Bacillus subtilis species, complex. Due to their gyrA and cheA sequences, the members of the B. subtilis species complex were identified as belonging to the type Bacillus atrophaeus. The plant growth-promoting effect was examined with Arabidopsis seedlings in potted experiments. Cold-tolerant members of the types Bacillus simplex, Bacillus atrophaeus and Bacillus pumilus showed a clear phytostimulatory effect (example 6). The B. simplex (ABI02S1), B. atrophaeus (ABI02A1), and B. pumilus (ABI02P1) strains were selected as examples for further examinations. These strains are described below:

Bacillus atrophaeus differs from B. subtilis through its DNA sequence and the formation of a dark pigment (Nakamura 1989). Its ability to promote plant growth has been documented (Karlidag et al. 2010, Ertuk et al. 2011, Chan et al. 2013). The taxonomic affiliation of the strain ABI021A1 with Bacillus atrophaeus was determined by comparing its 16S rRNA, qyrA and cheA partial sequence (see example 1):