Biocontrol mechanisms of Streptomyces sp. HU2014 against Rhizoctonia spp. caused wheat sharp eyespot

Abstract

Wheat is the important source of global food and its yield is affected by several main factors, such as diseases, pests, weeds and climates. Wheat sharp eyespot (WSE) is a serious disease caused by the phytopathogens Rhizoctonia cerealis and R. solani. R. cerealis has a higher pathogenicity than R. solani and is the main pathogen of WSE in China. Currently, the principal method of controlling this disease is by the use of synthetic chemical fungicides. In order to develop sustainable agriculture and protect the ecological environment, biocontrol approach is a possible alternative, which uses beneficial microorganisms to control pathogens and improve plant health. Streptomyces spp. belong to these microbes and are researched in the plant protection field. Some species in the genus Streptomyces have been identified as potential biocontrol agents against phytopathogens. However, reports on using Streptomyces spp. against R. cerealis are lacking. A new strain of Streptomyces sp. HU2014 was investigated to gain insight into the determinants of its utility as a potential biocontrol agent. The main directions of this study were to evaluate the antifungal mechanisms of HU2014 against WSE and the ability to promote plant growth. The dissertation work provides a theoretical foundation and practical solution for screening biocontrol in agriculture. Previous work showed that the strain HU2014 had strong antifungal activities against phytopathogens. To further study this strain, Genome sequencing and average nucleotide identity (ANI) calculation of it were established using the Oxford Nanopore Technologies standard protocol and the J Species WS Online Service. The genome size of HU2014 was 8,170,612 bp and the values of ANIb and ANIm were 93.30% and 94.01% respectively. The result indicated that HU2014 is a novel Streptomyces species most closely related to Streptomyces albireticuli. The complete genome sequence of HU2014 has been deposited in GenBank under the accession number CP097123. The antifungal activities of HU2014 and its four fractions (F2, F4, F6 and F8) against R. cerealis G11 and R. solani YL-3 were conducted in the co-culture trial. The inhibition of mycelial growth by HU2014 against R. solani YL-3 and R. cerealis G11 were 90.3% and 63.9%, respectively. The aerial hyphae of the two phytopathogenic fungi was hollow, and the cell wall of them was damaged for observation under a scanning electron microscope. F6 and F8 fractions had the maximum inhibition rates of 100% to R. solani YL-3 and R. cerealis G11, followed by F4 and F2 (less than 60.50%). Spectrophotometric analysis was found that F6 and F8 had the same strong characteristic absorption peaks at 319 nm, 333 nm, and 351 nm. The main active components in the filtrated supernatants were determined to enrich in F6 and F8. Some active metabolites produced by HU2014 have bacteriostatic effect on pathogens. Ultra-performance liquid chromatography-mass spectrometry on the four extracts from the extracellular filtrate (EF) of HU2014 identified 10 chemical constituents in the Natural Products Atlas with high match levels (more than 90%). At the beginning of the study of the induce resistance of wheat to phytopathogens, the soil in pots for growing Bainong 4199 wheat was treated with different concentrations of extracellular extract (EF) HU2014 without fungi. Protective reactions of plants were noted by changes in the activity of enzymes. The activity of peroxidase (POD) and phenylalanine ammonia lyase (PAL) could be significantly induced by EF treatment at low concentrations, while the activity of polyphenol oxidase (PPO) had no significant difference compared to the control. A continuation of the induce resistance study of wheat supplemented with R. cerealis G11 showed that POD, PAL and β-1,3-glucanase (GLU) activity in wheat leaves with three different treatments were significantly increased compared to the untreated control at different time points. The induce resistance in wheat varieties Aikang 58 and Bainong 307 was mainly activated by diluted EF, and in Zhoumai 22 by R. cerealis G11. Concurrently, six specific gene expression analyses were performed for Aikang 58, Bainong 307, and Zhoumai 22 at each sampling hour post-inoculation with 500- fold diluted EF of HU2014 using QRT-PCR. The expression of PR1, PR2, PR3, PR5, PAL, and LOX were markedly induced. The strain HU2014 had an allelopathic effect on wheat and green bristlegrass. Four fractions in three concentrations were investigated. Seed treatment had a negative allelopathic effect on seedling height and root length of the two tested plants, except for F2 at a concentration of 1 mg/ml, which increased wheat seedling height. Promoting plant-growth has indirect effect on controlling wheat sharp eyespot. The test of promoting plant growth was established in vitro and pots. The results demonstrated that HU2014 can produce indoleacetic acid, siderophores, extracellular enzymes, and solubilized phosphate, and it can promote plant growth. The shoot height increased significantly by 12.9% to 22.4% in comparison to the non-inoculated control in the three cultivars at 28 days post-inoculation. Similarly, HU2014 was able to improve shoot dry weight in comparison to the non-inoculated control. The chlorophyll content increased by 27.3% to 87.1% compared with the non-inoculated control. The root dry weight increased significantly by 45.5% to 83.4% in comparison to the non inoculated control at 60 days post-inoculation. The colonization of an exogenous microorganism and the synergistic effect of it with other rhizosphere microbes are important, which affect the antibacterial and promoting growth functions of this microorganism. The effect of HU2014 inoculation on soil and native soil microorganisms was investigated. From the results, we found that HU2014 increased the malondialdehyde content of wheat leave, while decreased catalase activity, peroxidase activity. With HU2014 inoculation, the concentrations of total nitrogen, nitrate nitrogen, total phosphorus, and Olsen-phosphorus in the wheat rhizosphere increased. The diversity of rhizosphere bacteria, but not fungi clearly decreased by HU2014 inoculation. The compositions of bacterial and fungal community differed after HU2014 inoculation with Proteobacteria, Acidobacteriota, and Ascomycota being the dominant phyla in all treatments. In order to optimize the environmental and nutritional parameters for improving the production of antifungal components by HU2014 through statistical approaches, five factors of fermentation condition, carbon and nitrogen sources, and inorganic salt were investigated. The optimized fermentation conditions and medium components with shake-flask were as follows: incubation time, 10 days; temperature, 25°C; agitation speed, 150 rpm; inoculum size, 5% (v/v); initial pH, 7.0; dextrin 39.59 g/L, yeast extract 8.52 g/L, and KNO3 2.49 g/L. At last, we investigated the biocontrol ability of HU2014 against WSE in field, the physiological parameters, as well as characteristic traits in promoting growth were assessed. In 2022 and 2023, the most promoting growth indicators after HU2014 inoculation showed increasing, but the amplification of them in field was less than in laboratory conditions.