Exploiting Natural Compounds of Cenchrus Pennisetiformis Against Fusarium Wilt of Tomato Under Charomium Stress

Abstract

Tomato (Solanum lycopersicum L.) is the second most consumable vegetable worldwide cultivated around the globe and constitutes a major agricultural industry. Pakistan occupies 35th position in tomato production on the globe. Average yield in the country is low due to various biotic and abiotic constraints. Fusarium wilt disease caused by a soil-borne fungus Fusarium oxysporum f. sp. lycopersici Snyder & Hansen is amongst the most important and potentially destructive biotic issues for tomato crop throughout the world. Besides, toxic level of heavy metal like chromium (Cr) in agricultural soil has become worldwide public health trepidation due to its extensive use in several anthropogenic and consequent contamination of soil and water has become a serious concern. Its two oxidation states viz. Cr(III) and Cr(VI) are notorious owing to wide occurrence in the environment. Utilization of metal contaminated wastewater and soil to grow vegetables is a very common practice in Pakistan. Up till now, strategies have been adopted either for the management of pathogen or for metal, however, the management options under simultaneous action of both stresses need to be addressed. The current study was planned to manage Fusarium wilt of tomato under abiotic stress of chromium through exploiting natural compounds of an allelopathic grass, Cenchrus pennisetiformis (Hochst. & Steud.) Wipff.  Initially, in vitro bioassays were performed to assess tolerance levels of F. oxysporum f. sp. lycopersici and tomato seedling against various concentrations of Cr(III) and Cr(VI). Results exhibited significant reduction of 10–60% and 1080%, respectively in fungal biomass due to Cr(III) and Cr(VI) over range of 5350 ppm, and no growth beyond 350 ppm. Likewise, germination and seeding growth of tomato were declined by 10-90% due to 20-350 ppm solutions of Cr(III) and Cr(VI), and no growth was observed above concentration of 350 ppm.  Toxicity evaluation of culture filtrates and conidial suspension of F. oxysporum f. sp. lycopersici on germination and seedling growth of tomato under six different concentrations (50-300 ppm) of Cr(III) and Cr(VI) indicted the highest reduction (50-90%) in germination, seedling length and biomass due to culture filtrates. F. oxysporum f. sp. lycopersici conidial suspension or culture filtrates in combination with either Cr(III) or Cr(VI) showed less or parallel reduction to those obtained over corresponding fungal inoculum type or metal ions concentration alone.  Screening of different parts of C. pennisetiformis for their antifungal activity against F. oxysporum f. sp. lycopersici showed 40-88%, 16-92% and 19-94% reduction in fungal biomass due to various concentrations (1-6%) of methanolic leaf, stem and root extracts, respectively. Combined application of methanolic extracts and aqueous solutions of Cr(III) or Cr(VI) proved more inhibitory than either alone.  Methanolic shoot (leaf + stem) and root extracts were fractionated using four organic solvents viz. n-hexane, chloroform, ethyl acetate and n-butanol. Various concentrations (1.56-12.5 mg mL-1) of ethyl acetate sub-fractions of both the extracts proved the most effective both in the presence or absence of Cr(III) and Cr(VI) exhibited up to 100% inhibition in growth of the target fungal pathogen.  GC-MS analysis of ethyl acetate sub-fraction of methanolic shoot and root extracts confirmed the presence of phenols in the highest amount.  On the bases of results obtained in various laboratory bioassays, pot experiments were designed to assess the effect of multilateral interaction of host-pathogenmetal-soil amendment on disease severity, plant growth, protein-profiling by SDS-PAGE, and metal uptake by tomato plants. Soil was spiked with three concentrations (200, 300 and 400 ppm) of each of Cr(III) and Cr(VI), inoculated with conidial suspensions of F. oxysporum f. sp. lycopersici, and amended with 1% and 2% plant biomass of C. pennisetiformis. The highest reduction in dry biomass (70-90%) of tomato plant were observed due to biotic stress of F. oxysporum f. sp. lycopersici. Plant dry biomass was gradually deceased by 2090% and 60-90% and metal toxicity symptoms were intensified too with increase in concentrations (200-400 ppm) of Cr(III) and Cr(VI), respectively. Combined effect of F. oxysporum f. sp. lycopersici and Cr(III) was less drastic on plants, but plant mortality rate was increased as compared to either stress alone. However, for Cr(VI) the effect was more drastic especially at 300 and 400 ppm of the metal ions combined with pathogen resulting in the highest mortality (70%). Tomato plants exhibited more tendency to uptake Cr(VI) than Cr(III). Root exhibited higher capacity to accumulate metal than stem and leaf. Soil amendments with 2% biomass of C. pennisetiformis showed more remarkable results in decreasing both biotic and abiotic stresses by improving plant growth. However, the effect of soil amendment was more pronounced at lower concentrations (200 and 300 ppm) of metal ions. Changes observed through protein-profiles were well-linked with growth assays.  Differential level of relative expression were recorded for plant chitinase as well as metallothionein genes in tomato leaf due to the effect of soil amendment with 2% biomass of C. pennisetiformis on wilt disease caused by F. oxysporum f. sp. lycopersici under 400 ppm of Cr(III) and Cr(VI). Both biotic and abiotic stresses caused change in metabolic reaction of plants with up or down regulation of protein, and catalase and peroxidase activities. FTIR spectra of tomato leaf showed major changes in region of protein (amides I and II), lipid and carbohydrate. It was concluded that soil amendment with 2% plant biomass of C. pennisetiformis could be used to manage Fusarium wilt disease in tomato under Cr stress within concentration range of 200-300 ppm. This study would be milestone towards solution to intricate environmental problem caused by devastating fungal pathogen and carcinogenic heavy metals.