SELECTION AND USE OF COLD-TOLERANT BACILLUS STRAINS AS BIOLOGICAL PHYTOSTIMULATORS

Abstract

The invention relates to a biological means for improving the yield of cultivated plants. The areas of application of the invention are agriculture, horticulture and plant protection.

Claims

1-11. (canceled)

12. A composition for stimulating the growth of cultivated plants, comprising a cold-tolerant Bacillus strain, wherein the cold-tolerant Bacillus strain comprises a Bacillus atrophaeus species or a Bacillus simplex species or a mixture thereof, and wherein the cold-tolerant Bacillus simplex species comprises ABI02S1 DSM 32020 or ABI12 DSM 32283, or a mixture thereof.

13. The composition according to claim 12, wherein the cold-tolerant Bacillus strain comprises a Bacillus atrophaeus species, and wherein the Bacillus atrophaeus species comprises ABI02A1 DSM 32019, ABI03 DSM 32285, or ABI05 DSM 29418, or a mixture thereof.

14. The composition according to claim 12, wherein the cold-tolerant Bacillus strain comprises a cold-tolerant Bacillus strain comprising Bacillus atrophaeus ABIO2A1 DSM 32019, ABIO3 DSM 32285, or ABIOS DSM 29418, or a mixture thereof; and a cold-tolerant Bacillus strain comprising Bacillus simplex ABIO2S1 DSM 32020 or ABI12 DSM 32283, or a mixture thereof.

15. The composition according to claim 12, wherein the cold-tolerant Bacillus strain is formulated as a fluid spore suspension.

16. The composition according to claim 12, wherein the cold-tolerant Bacillus strain is used for treating seeds during planting or treating seeds after planting.

17. The composition according to claim 12, wherein the cold-tolerant Bacillus strain is formulated as a dry preparation (“dry stain”).

18. The composition according to claim 12, further comprising humic acid.

19. The composition according to claim 12, further comprising a mesophile plant growth promoting bacteria of the Bacillales group.

20. The composition according to claim 12, further comprising a plant growth promoting fungus Trichoderma sp.

21. The composition according to claim 15, wherein the fluid spore suspension comprises at least 2×10.sup.9 spores/ml.

22. The composition according to claim 15, wherein the fluid spore suspension comprises at least 1×10.sup.10 spores/ml.

23. The composition according to claim 16, wherein said treating is by pour administration or by spray administration.

24. The composition according to claim 17, wherein the cold-tolerant Bacillus strain is formulated with at least 5×10.sup.9 spores/ml.

25. The composition according to claim 17, wherein the cold-tolerant Bacillus strain is formulated with at least 2×10.sup.10 spores/ml.

26. The composition according to claim 19, wherein said mesophile plant growth promoting bacteria of the Bacillales group comprises a Bacillus bacteria or a Paenibacillus bacteria.

27. The composition according to claim 26, wherein said Bacillus bacteria comprises a Bacillus amyloliquefaciens ssp. plantarum.

Description

LEGEND FOR THE FIGURES

[0047] FIG. 1: Comparison of the growth of B. atrophaeus ABI02A1 and the mesophile strain FZB42 at 20° C. and 25° C.

[0048] FIG. 2: Growth of B. atrophaeus ABI02A1 at different temperatures. The growth optimum was determined at 33-35° C.

[0049] FIG. 3: Phase contrast: Bacillus simplex ABI02S1. The distal, oval endospores are clearly visible in the non-swollen spore parent cells.

[0050] FIG. 4: Improved germination of Zea mays following incubation with B. simplex ABI02A1 (bottom row) compared to the control.

[0051] FIG. 5: Growth stimulation (in g fresh weight) of rabidopsis thaliana through spore suspensions of cold-tolerant Bacillus strains. In comparison: The effect of B. amyloliquefaciens FZB42. Control: without inoculation of Bacillus spores. The columns show the average value of three independent tests.

[0052] FIG. 6: Stimulation of root growth of Arabidopsis thaliana by B. atrophaeus ABI02A1 (upper left), and B. simplex ABI02S1 (upper right). CK=Control without inoculation with bacteria

[0053] FIG. 7: Potted test with potato plants (Kürzinger GmbH 2011). Comparison of the tuber yield with untreated control. Application of 1 spore suspension per ha of FZB42 and B. pumilus ABI02P1. The spore suspension of B. simplex ABI02S1 showed a lower concentration by a factor of 5, and was applied in a correspondingly higher quantity (5 l/ha) to the potato plants.

[0054] FIG. 8: Application of B. pumilus ABI02P1 (1 L/ha) and B. simplex ABI02S1 to potato plants. Single application through staining with the spore suspension (FZB42 and Bacillus pumilus ABI02P1: 2×10.sup.19 cfu/ml, Bacillus simplex ABI02S1: 4×10.sup.9 cfu/ml) before planting.

[0055] FIG. 9: Randomised location map: 1 (control, no addition), 2 chemical fungicide (Monceren Pro, 1.5 l/ha), 3 FZB42 (0.5 l/ha), 4 FZB42 (1.0 l/ha), 5 FZB42 (2×0.5 l/ha), 8 ABI02A (1 l/ha). For each variant, four repetitions were conducted. The lot size was as follows: 6.90 m×3.00 m=20.70 m.sup.2, space between rows: 75 cm, space between tubers in the row: 30 cm. “Staining” of the tubers in the storage building: 0.5.2013, date planted: 06.05.2013, emergence date: 02.06. 2013, harvest: 30.08.2013, treatment: 07. 10. 2013.

[0056] FIG. 10: Reduction in disease symptoms (“black scurf”) following application of chemical fungicides (Monoceren Pro) and organic stimulators. The use of ABI02A ‘Bacillus atrophaeus’ (1.0 l/ha) leads to a considerable reduction in disease symptoms.

[0057] FIG. 11: The use of Monoceren (chemical fungicide) and spore suspensions of FZB42 and ABI02A1 (Bacillus atrophaeus) among potatoes of type Verdi (Sanitz, Kürzinger 2013) leads to an increase in yield (%). Quantities used: 1.0=1.0 kg/ha.

[0058] FIG. 12: The application of Bacillus atrophaeus ABI02A1 leads to a considerable increase in yield, which can be compared to that of the chemical fungicide Monoceren. With the mesophile strain FZB42, a somewhat higher yield increase was achieved. Quantities of fungicides used: 1.5 l/ha (Monceren), 0.5 1.0 l/ha and 2×0.5 I/ha with spore suspensions of FZB42 and ABI02A1.

[0059] FIG. 13: At the same time, the infestation of sclerotia (Rhizoctonia solani) was reduced with the application of Bacillus atrophaeus ABI02A1 compared to the untreated control. Illustration of all infestation classes (% sclerotia infestation) with the use of Monoceren, FZB42 and B. atrophaeus. B. atrophaeus leads to a reduction in sclerotia infestation compared to the untreated control. Infestation classes: 0, <1, 1.1-5, 5.1-10, 10.1-15 and >15.

LIST OF REFERENCES

[0060] Abd El-Rahman, H. A., Fritze, D., Cathrin Sproer, C., Claus, D. 2002. Two novel psychrotolerant species, Bacillus psychrotolerans sp. nov. and Bacillus psychrodurans sp. nov., which contain ornithine in their cell walls. Int. J. Syst. Evol. Microbiol. 52, 2127-2133

[0061] Bai, Y., D'Aoust, F., Smith, D. L., Driscoll, B. T. 2002. Isolation of plant-growth-promoting Bacillus strains from soybean root nodules. Can. J. Microbiol. 48, 230-238 (2002)

[0062] Barka, A. E., Nowk, J., Clement, C. 2006. Enhancement of chilling resistance of inoculated grapevine plantlets with plant growth promoting rhizobacteria Burkholderia phytofermans strain PsJN. Appl Environ Microbiol 72:7246-7252

[0063] Benhamou, N., Kloepper, J. W., Quadt-Hallman, A., Tuzun, S. 1996. Induction of defense-related ultrastructural modifications in pea root tissues inoculated with endophytic bacteria. Plant Physiol. 112, 919-929

[0064] Borriss, R. 2011. Use of plant-associated Bacillus strains as biofertilizers and biocontrol agents, In: Maheshwari DK (ed). Bacteria in agrobiology: plant growth responses. Springer, Germany, pp 41-76

[0065] Chan, W. Y., Dietel, K., Lapa, S. et al. 2013. Draft Genome Sequence of Bacillus atrophaeus UCMB-5137, a Plant Growth-Promoting Rhizobacterium. Genome Announcement 1, e00233-13

[0066] Erturk, Y., Cakmakci, R., Omur Duyar, O., Turan, M. 2011. The Effects of Plant Growth Promotion Rhizobacteria on Vegetative Growth and Leaf Nutrient Contents of Hazelnut Seedlings (Turkish hazelnut cv, Tombul and Sivri). Int. J. Soil Science, 6, 188-198.

[0067] Feller, G. & Gerday C. 2003. Psychrophilic enzymes: hot topics in cold adaptation. Nature Reviews Microbiology 1, 200-208

[0068] Gulati, A., Vyas, P., Rai, P., Kasana, R. C. 2009. Plant growth promoting and rhizosphere-competent Acinetobacter rhizosphaerae strain BIHB 723 from the cold deserts of the Himalayas. Curr. Microbiol. 58, 371-377

[0069] Handelsman, J., Raffel, S., Mester, E.H. et al. 1990. Biological control of damping-off of alfalfa seedlings with Bacillus-cereus UW85. Appl. Env. Microbiol. 56, 713-718

[0070] Katiyar, V., Goel, R. 2003. Solubilization of inorganic phosphate and plant growth promotion by cold tolerant mutants of Pseudomonas fluorescens. Microbiol Res

[0071] Idriss, E. E., Makarewicz, O., Farouk, A. et al. 2002. Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant -growth-promoting effect. Microbiology 148, 2097-2109.

[0072] Kaushik, R., Saxena, A. K., Tilak, K. V. B. R. 2002. Can Azospirillum strains capable of growing at a suboptimal temperature perform better in field-grown-wheat rhizosphere. Biol Fertil Soils 35, 92-95

[0073] Karlidag, H., Esitken, A., Yildrim, E. et al. 2010. Effects of plant growth promoting bacteria on yield, growth, leaf water content, membrane permeability, and ionic composition of strawberry under saline conditions. J. Plant Nutr. 34, 34-45

[0074] Krishnamurti, S., Ruckmani, A., Pukall, R., Chakrabarti, T. 2010. Psychrobacillus gen. nov. and proposal for reclassification of Bacillus insolitus Larkin & Stokes, 1967,

[0075] B. psychrotolerans Abd-El Rahman et al., 2002 and B. psychrodurans Abd-El Rahman et al., 2002 as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrotolerans comb. nov. and Psychrobacillus psychrodurans comb. nov. Syst. Appl. Microbiol. 33, 367-373

[0076] Kumar, P. K., Joshi, P., Bisht, J. K. et al. 2011. Cold-Tolerant Agriculturally Important Microorganisms. In: Plant Growth and Health Promoting Bacteria. Microbiology Monographs. Volume 18, 2011, pp 273-296.

[0077] Lopez-Bucio, J., Campos-Cuevas, J. C., Hernandez-Calderon, E. et al. 2007. Bacillus megaterium rhizobacteria promote growth and alter root-system architecture through an auxin-and ethylene-independent signaling mechanism in Arabidopsis thaliana. Mol. Plant Microbe Interact. 20, 207-217

[0078] Mishra, P. K., Bisht, S. C., Bisht, J. K., Bhatt, J.C. 2012. Cold-tolerant PGPRs as bioinoculants for stress management. In: Maheshwari DK (ed). Bacteria in agrobiology: Stress Management, DOI 10.1007/978-3-642-23465-1_6, #Springer-Verlag Berlin Heidelberg

[0079] Nakamura, L. K. 1989. Taxonomic relationship of black-pigmented Bacillus subtilis strains and a proposal for Bacillus atrophaeus sp. nov. Int. J. Syst. Bacteriol. 39, 295-300.

[0080] Prevost D, Drouin P, Laberge S, et al. 2003. Cold-adapted rhizobia for nitrogen fixation in temperate regions. Can. J. Bot. 81, 1153-1161

[0081] Schwartz, A. R., Ortiz, I., Maymon, M. et al. 2013. Bacillus simplex—A little known PGPB with anti-fungal activity—alters pea legume root architecture and nodule morphology when coinculated with Rhizobium leguminosarum by. viciae. Agronomy 3, 595-620

[0082] Selvakumar, G., Kundu, S., Joshi, P. et al. 2008a. Characterization of a cold-tolerant plant growth-promoting bacterium Pantoea dispersa 1A isolated from a sub-alpine soil in the North Western Indian Himalayas. World J. Microbiol. Biotechnol. 24, 955-960

[0083] Selvakumar, G., Mohan, M., Kundu, S. et al. 2008b. Cold tolerance and plant growth promotion potential of Serratia marcescens strain SRM (MTCC 8708) isolated from flowers of summer squash (Cucurbita pepo). Lett. Appl. Microbiol. 46:171-175

[0084] Zhang, H., Prithiviraj, B., Charles, T. C. et al. 2003. Low temperature tolerant Bradyrhizobium japonicum strains allowing improved nodulation and nitrogen fixation of soybean in a short season (cool spring) area. Eur. J. Agron. 19,205-213