A bacterial endophyte isolated from wheat seeds showed the ability to colonize root and shoot tissues during the initial establishment of seedlings. The isolate, named JunSE1L, belongs to the species Bacillus atrophaeus. In laboratory tests in the United States, it also inhibited the growth of Fusarium proliferatum and Mucor hiemalis, two fungi used as targets in the study.
The research evaluated winter wheat seeds, cultivar Juniper. Scientists sterilized the surface of the seeds with ten percent hydrogen peroxide. Then, they incubated the material in LB medium with two percent agar, at 22 degrees Celsius. White colonies appeared on 44 out of 55 seeds, a proportion close to 80 percent.
The team purified two types of colonies. The work focused the analysis on the larger, rougher form, called JunSE1L. Identification by sequencing the 16S rRNA gene confirmed 100 percent identity with Bacillus atrophaeus.
The isolate appeared in different plant compartments after six days of growth in sterilized sand. Researchers recovered colonies in soil adhering to roots, on the root surface, in root segments, and in leaf and shoot tissues. Emergence occurred most frequently at the cut edges of tissues. This observation indicated mobilization of the endophyte from the seed during germination.
JunSE1L exhibited strong plasticity depending on the culture medium. In LB medium, it formed compact, rough, and hydrophobic colonies. Water droplets remained nearly spherical on the colony surface for about one and a half to two hours. In minimal medium, the colonies spread out more, became less hydrophobic, and absorbed the droplet in about ten minutes.
The behavior also changed in spore formation. In minimal medium, spores appeared inside the mother cells on the fifth day. Free spores emerged on the seventh day. In LB, sporulation took longer and only appeared after 15 days.
The bacteria formed a biofilm in a rich medium. In static liquid cultures with LB, JunSE1L produced a thick film at the air-liquid interface in 48 hours. In minimal medium, under the same conditions, no film was visible. This result indicated a direct influence of nutrient availability on the lifestyle associated with surfaces.
The isolate produced compounds with surface activity. During growth in minimal liquid medium, the surface tension of the filtrate dropped from 72,2 to approximately 30 millinewtons per meter. The drop occurred during the late exponential growth phase. The estimated critical micelle concentration for the precipitated fraction at pH two reached approximately 0,125 milligrams per milliliter.
JunSE1L also exhibited extracellular activities associated with microbial competition. Colonies formed hemolysis halos on blood agar after three days. On skim milk agar, they formed casein hydrolysis zones, a sign of protease secretion. The isolate did not show detectable phosphate solubilization on Pikovskaya agar. It also did not indicate nitrogen fixation in nitrogen-free medium.
In antifungal assays with live cells, a ten-microliter bacterial suspension generated zones of inhibition against both fungi. The median halo reached 3,0 centimeters for Mucor hiemalis and 2,0 centimeters for Fusarium proliferatum. Control treatments with sterile distilled water did not inhibit fungal growth.
The cell-free supernatant maintained activity only against Mucor hiemalis under the tested conditions, filter paper discs soaked in the supernatant produced an average halo of 1,5 centimeters against this fungus. The same supernatant did not generate detectable inhibition against Fusarium proliferatum. The scientists state in the study: the effect depended on the assay design and the fungal species. The biochemical mechanism involved was not identified.
The application of JunSE1L to the surface of seeds also showed dose dependence. Concentrations of 10⁴ and 10⁵ colony-forming units per milliliter resulted in little or no recovery of the bacteria in the seed. A concentration of 10⁸ colony-forming units per milliliter allowed the recovery of approximately 10⁵ colony-forming units per seed. At 10⁶ colony-forming units per milliliter, the recovery was close to 10² colony-forming units per seed.
The seeds showed radicle emergence at all doses tested. However, the scientists did not measure root length, shoot height, or dry biomass. Therefore, the study did not confirm an effect of the isolate on seedling vigor. The results indicate the potential of JunSE1L as a model for studying native endophytes of wheat seeds and their contribution to the initial assembly of the plant microbiome.
More information at doi.org/10.3390/seeds5030030
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