Co-culture of Pseudomonas chlororaphis and Saccharomyces cerevisiae to create a complex biological product

Hnologies for the production of new biological preparations that contain plant growth stimulating lines of bacteria (Plant Growth Promoting Bacteria, PGPB) or fungi (Plant Growth Promoting Fungi, PGPF). Fungi and bacteria co-inhabit the rhizosphere of higher plants and fungal-bacterial interactions permanently occur. In recent years, biological preparations based on bacteria and fungi (insecticide Biostop, fungicide Sporobacterin) have entered the agrochemical and pesticide market. However, special requirements of bacteria and fungi for nutrient media, aeration, and culture regimes imped production of combined bacterial-fungal biologicals. In this work, we have established for the first time the positive effect of Saccharomyces cerevisiae Y-4317 on the growth of Pseudomonas chlororaphis subsp. auerofaciens B-5326 and revealed the optimal regimes and the composition of the nutrient medium for co-culture of microorganisms of different taxonomic groups (PGPB and PGPF). The high efficiency of using sugar beet molasses for co-culture of P. chlororaphis and S. cerevisiae has been shown. Our findings revealed the stimulating effect of the liquid culture (LC) on the seed germination energy and germination rate of cereals. The aim of this work was to develop a protocol for co-culture of bacteria Pseudomonas chlororaphis subsp. auerofaciens B-5326 and yeast Saccharomyces cerevisiae Y-4317 to create a biological preparation stimulating seed germination and initial growth of cereal plants. Lyophilized strains P. chlororaphis and S. cerevisiae were obtained from the All-Russian Collection of Industrial Microorganisms of the National Research Center Kurchatov Institute - GosNIIgenetica (Moscow). Seeds of maize ( Zea mays L.) hybrid Delitop, wheat ( Triticum aestivum L.) variety Mironovskaya 808, barley ( Нordeum vulgare L.) variety Scepter served for testing bioactivity of the biological. After restoring viability, the strains were cultured for 48 hours on a shaker SPH-2102 (BIORUS, Belarus) using three nutrient media differing in the carbon source (glucose, fructose, and sugar beet molasses). Microbial growth (colony-forming units, CFU) and biomass were assessed during co-culture (from 0 to 72 h) on the shaker and in a lab fermenter BIORUS GJ (BIORUS, Belarus) at different temperatures (from 20 to 32 °С) and airflow rates (from 1 to 6 L/h). The seeds were treated by spraying with 1:200-1:25 serial dilutions of the liquid co-culture. After 12 h, the seeds were placed into Petri dishes with water. The germination energy and seed germination rate were determined in 3 and 7 days, respectively. The research data showed that media containing sugar beet molasses completely satisfies the need of co-cultured P. chlororaphis and S. cerevisiae for basic nutrients, and the titers did not fall below 6×108 and 3×106 CFU/ml, respectively. The total biomass was 20.4 g/l, or 17-22 % higher than on the media with glucose or fructose. For co-culture, the optimal conditions were 30°C, aeration mode 4 l/h and 24 h of growth. A positive effect of S. cerevisiae on the viability of P. chlororaphis during 72 h co-culture was demonstrated. The abundance of P. chlororaphis in the co-culture with S. cerevisiae was 1½106 CFU/ml vs. 5½104 CFU/ml in pure culture of P. chlororaphis . Probably, a higher viability of P. chlororaphis and stimulation of its growth is due to phytohormones produced by S. cerevisiae during co-culture. Preliminary testing revealed stimulating effect of the biological on the germination energy and seed germination rate for barley, wheat and corn. All serial dilutions of the liquid co-culture exhibited a clear trend towards an increase in seed germination in the cereals tested. The maize seed germination was most stimulated. A 1:200 dilution of the biological led to the maximum increase in seed germination of the crops. Our research data identify the key parameters of the co-culture of PGPB P. chlororaphis and PGPF S. cerevisiae and thereby create the prerequisites for the development of a biological based on microorganisms of vadifferent taxonomic groups.