In the germination process of wheat seeds, a bluish-purple pigment violacein, a bisindole pigment produced by Gram-negative bacteria was detected. Violacein has a wide range of physiological activities such as antibacterial activity, antitumor activity, and antioxidant activity have been reported. We identified this bluish-purple pigment produced in the wheat roots with symbiosis of a rhizobacteria constructing a consortium of microorganisms show antifungal effect to plant diseases, such as Fusarium spp.

Two Japanese bread wheat cv. Norin 61 and Yumeshiho, one Afghan landrace #654 were cultivated with pre-treatment of Duckweed fermented fertilizer (DWF) including 1 μM KODA (9-hydroxy-10-oxo-12(Z), 15(Z)-octadecadienoic acid) as a biostimulant (Haque et al., 2016) for an experiment of arbuscular mycorrhizal symbiosis of Rhizophagus irregularis (MYCOGEL®, Hyponex) with in the winter season of 2019-20. The harvested seeds were sowed without seed sterilization in a petri dish in January 2021, keeping in low temperature condition to germinate at 4 °C for 2 weeks, then greening at room temperature (around 20 °C) and blue-purple pigment detected with seed transmission of the symbiotic bmicroorganisms. When the extract obtained by extracting the potato semi-synthetic agar medium with methanol was analyzed by LC-MS, violacein (MW = 343.34) and deoxyviolacein (MW = 327.34) were identified as blue-purple pigments. When the extract was solvent-fractionated with water and ethyl acetate, a red-pink pigment was detected in the aqueous layer fraction produced.

The colonies were isolated from the germinated seeds of Norin 61 and cultured in King's B medium, and it was found to be a bacterial population of multiple species. The isolated colonies of bacteria produce violacein/decxyviolacein were Janthinobacterium lividum identified with 16S rRNA amplicon sequence. Low temperature condition for germination at 4-6 °C after D-KODA/DWF treatment on wheat dry seeds showed remarkable seminal roots elongation of cv. Norin 61 but Afghan landrace # 654. Symbiotic rhizobacteria such as Janthinobacterium and Duganella which produce violacein and Chryseobacterium, Pantoea were found in Norin 61, but Massilia was detected relatively often in Afghan landrace #654.

They showed slight inhibition of hyphal growth of Fusarium fungus of Fusarium head blight, and some showed high antifungal activity. We have found that biostimulant treatment of wheat modifies the rhizosphere flora and may tune the rhizosphere environment for seed root elongation and antifungal activity.

The growth of seedlings was promoted by inoculation with a cocktail of isolated rhizosphere bacteria, and the volume of the root system was particularly increased with the effect of D-KODA/DWF on the moisture damage. Occasionally, Erwinia persicina produce the aqueous red-pink pigment causes red grains of barley was detected in the rhizosphere. We will clarify the effects of these non-pathogenic and consortium formation with other rhizosphere bacteria and the growth promoting effect of the wheat root system.