Cellulolytic bacteria and association of effective microorganisms for biocontrol of root rot infections in sugar beet (Beta vulgaris L.)

At the present time, many farmers growing sugar beet ( Beta vulgaris L.) reduce application of fertilizers and crop rotations, which leads to accumulation of phytopathogens. The main pathogens of sugar beet causing root rot are fungi from genera Fusarium Lk.:Fr. and Alternaria (Fr.) Keissi. Chemical fungicides are used worldwide to protect crops but plant pathogenic fungi acquire resistance against conventional chemicals. Therefore, the biological methods of plant protection are relevant. In our previous works, we have designed the association of effective microorganisms (EM Association) which includes nitrogen-fixing Azotobacter chroococcum and phosphate-mobilizing Bacillus megaterium bacteria. This association can increase the productivity of sugar beet plants but does not possess antifungal activity against sugar beet root rot. Cellulolytic bacteria are an important component of microbiocenoses. They play a significant role in soil processes; their number is an indicator of soil fertility and ecological quality. Colonizing rhizosphere of plants, they synthesize bioactive substances, including antifungal metabolites. Among cellulolytic bacteria there are active antagonists of fungal root rot causative agents. In this paper we present our research findings on the antifungal properties of a new cellulolytic strain Bacillus sp. C-82/3 and the first effective association of this strain with nitrogen-fixing and phosphate-mobilizing bacteria which promotes sugar beet growth and yield. The goal of the research was to evaluate antifungal activity of the novel strain of cellulolytic bacteria Bacillus sp. C-82/3 isolated from soil rhizosphere of healthy sugar beet plants in the South-East of Kazakhstan (Zhambyl region), to enrich the EM association developed with this strain, and to assess the plant growth promoting activity of the improved EM Association and its ability to biocontrol rot root infections under field condition. Antifungal activity was determined in agar block diffusion tests against Alternaria alternata (Fr.) Keissl, Fusarium solani (Mart.) Sacc. and F. oxysporum Schlecht. The strain was grown on the Hutchinson’s medium (1.0 g/l К2НРО4, 0.1 g/l CaCl2, 0.3 g/l MgS04, 2.5 g/l NaNO3, 1.0 g/l NaCl, 0.01 g/l FеСl3, 20 g/l wheat straw, 5,0 g/l yeast extract; pH 7.0). Blocks with growing culture were cut out, and put on Petri dishes with potato-dextrose agar earlier inoculated with fungi, and cultured at 28 °С for 3 days. Antifungal activity was assessed by the diameter of growth inhibition zone. To study the plant growth promoting activity of the EM association with Bacillus sp. C-82/3, the cv. Aisultan seeds were treated with the bacterial suspension (107 cells/ml) at 23 °С for 2 hours. The stem and root length measured in the inoculated seedling after 30-day growing in a climatic chamber (Constant Сlimate Сhamber HPP750, Memmert GmbH + Co. KG, Germany) were compared to the control. Field tests were conducted in the South-East of Kazakhstan (Zhambyl region, Kaiyndy farm) in 2017-2018. The results of lab screening showed high antifungal activity of the novel strain Bacillus sp. C-82/3 with the mean inhibition halos of 28.9±0.2 mm for F. oxysporum , 38.2±0.3 mm for F. solani , and 46.6±0.9 mm for A. alternate . The improved EM Association which includes three strains ( Bacillus megaterium , Azotobacter chroococcum and Bacillus sр. С-82/3) was characterized by high growth-promoting activity. Germination of the inoculated seeds was 7-16 % higher, and stem and root length increased 1.2-1.5-fold and 1.1-2.0-fold, respectively, as compared to control (р ≤ 0.05). We also revealed the high ability of the EM Association containing Bacillus sр. С-82/3 strain to suppress sugar beet root rot pathogens in soil biocenosis. Seed inoculation with the microbial association decreased the damage to seedlings 2.3 times, to roots 3.0 times. The yield of sugar beet was 34.2±2.3 c/ha higher compared to control (р ≤ 0.05). Thus, our data are the first evidence that the EM Association with Bacillus sp. C-82/3, a new cellulolytic strain with high antifungal activity that we have detected, is effective against root rot infection and promotes an increase in sugar beet yield under field condition.