Four new Swiss strains of Bacillus amyloliquefaciens

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Plant pathogens cause high losses in horticulture and agriculture. The extensive use of chemical products for many years has led to the loss of biological soil ...
Four new Swiss strains of Bacillus amyloliquefaciens  usable in biological fertilization. Rossi Jérémy, Crovadore Julien, Pelleteret Pegah, Cochard Bastien et Lefort François

Plant pathogens cause high losses in horticulture and agriculture. The extensive use of chemical products for many years has led to the loss of biological soil fertility, toxicity against non‐target organisms, accumulation of toxic molecules in ecosystems and the development of pesticide resistances. The reduction of authorized synthetic molecules and the decline of newly registered chemical pesticides concomitantly raised a growing interest for biological control and biological fertilization methods based on microorganisms. Bacillus amyloliquefaciens is a bacterium with a high potential of antagonism towards plant pathogens such as fungi and bacteria. This species is also considered as a plant growth inductor. Very few strains are commercially available. The present works aimed to characterize new strains with high commercial potential.

Results

Experiments Four Swiss strains of Bacillus amyloliquefaciens: P1 ‐ C2 ‐ BA3 ‐ BA4. were compared to the commercial strain IT45.

Figure 1. Electrophoresis gel for confirmation of strains identity.

The strains were identified by PCR amplification and DNA sequencing in order to confirm their identity (Figure 1). Various tests were carried out in order to characterize these strains:  Establishment of growth curves. Around 30 hours, the largest concentration of bacteria in the exponential growth is observed. Therefore strains were incubated for 30h under these conditions in most of the following experiments.  Resistance to UV rays (Figures 2 and 3). The IT45 strain is particularly more sensitive to UV while the BA3 strain has more resistant colonies with 15 minutes of exposure. BA3 could therefore be suitable for foliar applications.

10 min

15 min

Figure 2. 48h of C2 culture after 0, 5, 10 and 15min of UV exposition.

 Antibiogram. The five strains were particularly sensitive to cephalexin with a systematic inhibition at the lowest concentration, whereas no inhibition was measured for the sulfanilamide in the tested concentrations.

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Figure 3. UV exposition of the five B. amyloliquefaciens strains. « + » means uncountable colonies.

Botryosphaeria iberica growth faces to B.  amyloliquefaciens

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9 8 T (‐) P1 C2 IT45 BA3 BA4

7 6 Growth (cm)

 Antagonism In order to evaluate their antagonism potential, the strains were screened in vitro against a selection of 29 phytopathogenic fungi (Figures 4 and 5). The strain P1 was the most effective in 50% of cases. Bacillus amyloliquefaciens P1 and C2 were highly complementary and were antagonists at 93% for the 29 phytopathogenic micro‐organisms.

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Figure 5. Testing for antagonism potential. A. P1 against B. iberica B. BA3 against B. cinerea

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Figure 4. Growth curves of Botryosphaeria iberica faces to Bacillus amyloliquefaciens.

Growth induction Germination of corn salad seeds

In order to evaluate the potential of the growth induction, tests were carried out on carrot, lettuce and parsley plants in climatic chambers. A germination and seeding test was also carried out on corn salad seeds (Figure 6). In addition to slightly promoting the germination rate, the four strains of Bacillus amyloliquefaciens P1, C2, BA3 and BA4 increased the seeding of corn salad seeds.

Seeding of corn salad seeds

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Figure 6. Test of germination and seeding on corn salad seeds

Endophytic potential

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In order to evaluate the endophytic potential of the strains, these five strains were inoculated in 1 year apple trees, grown in greenhouse, using 2 methods : watering the bacterial solution into the substrate or soaking the plantlets into the bacterial solution (Figures 7 and 8). With both inoculation methods, the 5 strains of Bacillus amyloliquefaciens were found as endophytes in apple trees.

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Figure 7. A. Isolation of BA4 from a young leaf, after soaking B. Isolation of P1 and others endophytes from an apple tree with soaked stem.

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Figure 8. Presence of endophytes after soaking «t» or watering «a» in the different bacterial solution.

These In vitro trials of antagonism showed very promising results and allowed to characterize the P1 strain as the most effective in 50 % of the cases and its association with C2 could be highly efficient against phytopathogens. The effects as fertilizers and biological control agents were demonstrated. Thus, by reducing the germination and seedling times and by protecting against pathogenic microorganisms, the 4 new strains of B. amyloliquefaciens bear a real potential for agronomical applications. The endophytic behavior demonstrated for these strains could be an additional asset.

10th Annual Biocontrol Industry Meeting – ABIM 19 ‐ 21 October 2015 Basel, Switzerland