BIOTECHNOLOGICAL POTENTIAL OF BACTERIAL METABOLITES IMMOBILIZED ON IRRADIATED NANO CLAY PARTICLES IN RELATION TO CROP PRODUCTION

Abstract

Potato is one of the major food crops belonging to family Solanaceae. Black scurf and stem canker, caused by Rhizoctonia solani are destructive diseases of potato causing serious damage in most potato-producing areas throughout the world. Chemical pesticides are widely used to fungal pathogens control. However, the application of chemical pesticides is harmful to both the environment and human health. Therefore, the use of biocontrol agents (BCAs) is considered to be potentially sustainable, cost-effective and eco-friendly ways to control R. solani. Accordingly, 84 isolates of PGPR were recovered and screened for their ability to produce antifungal compound(s), from rhizosphere of healthy potato plant collected from different areas. One strain was selected based on its ability to suppress the growth of R. solani. Phylogenetic analysis of this strain based on 16S rRNA gene sequences showed highest similarity (99%) with Bacillus sp. GRP (GenBank accession number: MK030136). The Bacillus sp. GRP culture filtrate contained protease, diffusible antibiotic, hydrogen cyanide and siderophore, it was capable of inhibiting growth of the pathogen up to 15 days compared to 7 and 10 days for other Bacillus isolates. It also produced indole acetic acid which promoted plant growth. Morphological and structural changes that took place as a result of Bacillus sp. and R. solani interaction were evaluated using light, SEM and TEM. The results showed that Bacillus sp. caused loss of structural integrity, abnormal coiling, shriveling and lysis of the R. solani hyphae, in addition to complete cytoplasm and internal organelles depletion. Among four media tested, the maximum growth and antifungal activities were found in tryptone soya broth medium under static condition at 30°C, pH7, inoculum density of 1% and 72 h incubation. Antifungal substances were extracted and identified by Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FT-IR), Ultraviolet-visible spectroscopy (UV-Vis) and Liquid Chromatography Mass Spectrometry (LC-MS) that revealed the presence of two compounds which were assigned as lipopeptides (fengycin and surfactin). Significant inhibitory effect of lipopeptides was observed at concentration as low as 200 µg/ml. No changes in viability of human cell line were observed even at the highest concentration tested (200 µg/mL) as indicated by inverted microscope. Lipopeptides showed an increased stability to high temperature, pH, UV and sunlight. Nanoclay particles exposed to different doses of gamma irradiation (0, 25, 50 and 100 KGy) were studied. Bacillus sp. GRP and lipopeptides were immobilized on nanoclay to form a bionanocomposite and bio-nano-fungicide. Bionanocomposite and bio-nano-fungicide were characterized by Dynamic light Scattering Measurments (DLS), FTIR spectroscopy, X- ray diffraction (XRD), TEM and SEM. Bionanocomposite exhibited biocontrol efficiency along 8 months storage, while bio-nano-fungicide maintained higher activity during 12 months at room temperature in comparison with the free lipopeptide. Both in vitro and greenhouse experiments for both bioformulations showed high inhibition of R. solani radial growth. Results indicate that the prepared bionanocomposite and bio-nano-fungicide are a promising alternative to the commercial products.