Mohamed E. I. Badawy

A Biopolymer Chitosan and Its Derivatives as Promising Antimicrobial Agents against Plant Pathogens and Their Applications in Crop Protection

Recently, much attention has been paid to chitosan as a potential polysaccharide resource. Although several efforts have been reported to prepare functional derivatives of chitosan by chemical modifications, few attained their antimicrobial activity against plant pathogens. The present paper aims to present an overview of the antimicrobial effects, mechanisms, and applications of a biopolymer chitosan and its derivatives in crop protection. In addition, this paper takes a closer look at the physiochemical properties and chemical modifications of chitosan molecule. The recent growth in this field and the latest research papers published will be introduced and discussed.

Antimicrobial and inhibitory enzyme activity of N-(benzyl) and quaternary N-(benzyl) chitosan derivatives on plant pathogens.

Chemical modification of a biopolymer chitosan by introducing quaternary ammonium moieties into the polymer backbone enhances its antimicrobial activity. In the present study, a series of quaternary N-(benzyl) chitosan derivatives were synthesized and characterized by (1)H-NMR, FT-IR and UV spectroscopic techniques. The antimicrobial activity against crop-threatening bacteria Agrobacterium tumefaciens and Erwinia carotovora and fungi Botrytis cinerea, Botryodiplodia theobromae, Fusarium oxysporum and Phytophthora infestans were evaluated. The results proved that the grafting of benzyl moiety or quaternization of the derivatives onto chitosan molecule was successful in inhibiting the microbial growth. Moreover, increase water-solubility of the compounds by quaternization significantly increased the activity against bacteria and fungi. Exocellular enzymes including polygalacturonase (PGase), pectin-lyase (PLase), polyphenol oxidase (PPOase) and cellulase were also affected at 1000 mg/L. These compounds especially quaternary-based chitosan derivatives that have good inhibitory effect should be potentially used as antimicrobial agents in crop protection.

Synthesis and structure-activity relationship of N-(cinnamyl) chitosan analogs as antimicrobial agents.

The current study focuses on the preparation of new N-(cinnamyl) chitosan derivatives as antimicrobial agents against nine types of crop-threatening pathogens. Chitosan was reacted with a set of aromatic cinnamaldehyde analogs by reductive amination involving formation of the corresponding imines, followed by reduction with sodium borohydride to produce N-(cinnamyl) chitosan derivatives. The structural characterization was confirmed by (1)H and (13)C NMR spectroscopy and the degrees of substitution ranged from 0.08 to 0.28. The antibacterial activity was evaluated in vitro by minimum inhibitory concentration (MIC) against Agrobacterium tumefaciens and Erwinia carotovora. A higher inhibition activity was obtained by N-(α-methylcinnamyl) chitosan with MIC 1275 and 1025 mg/L against A. tumefaciens and E. carotovora, respectively followed by N-(o-methoxycinnamyl) chitosan (MIC=1925 and 1550 mg/L, respectively). The antifungal assessment was evaluated in vitro by mycelial radial growth technique against Alternaria alternata, Botrytis cinerea, Botryodiplodia theobromae, Fusarium oxysporum, Fusarium solani, Pythium debaryanum and Phytophthora infestans. N-(o-methoxycinnamyl) chitosan showed the highest antifungal activity among the tested compounds against the airborne fungi A. alternata, B. cinerea, Bd. theobromae and Ph. infestans with EC₅₀ of 672, 796, 980 and 636 mg/L, respectively. However, N-(p-N-dimethylaminocinnamyl) chitosan was the most active against the soil born fungi F. oxysporum, F. solani and P. debaryanum (EC50=411, 566 and 404 mg/L, respectively). On the other hand, the chitosan derivatives caused significant reduction in spore germination of A. alternata, B. cinerea, F. oxysporum and F. solani compared to chitosan and the reduction in spore germination was higher than that of the mycelia inhibition. The synthesis and characterization of new chitosan derivatives are ongoing in our laboratory aiming to obtain derivatives with higher antimicrobial activities and used as safe alternatives to harmful microbicides.

Biodegradation of Chlorpyrifos by a Newly Isolated Bacillus subtilis Strain, Y242

ABSTRACT A bacterial strain Y242 isolated from agricultural wastewater was found to be highly effective in degrading chlorpyrifos. On the basis of morphology, physiological characteristics, biochemical tests, and phylogenetic analysis of 16S rRNA sequence, the isolate was identified as Bacillus subtilis. The efficiency of the B. subtilis Y242 isolate as a chlorpyrifos degrader was examined under different culture conditions formulated according to the Plackett-Burman experimental design. It was observed that B. subtilis Y242 was able to utilize chlorpyrifos as a sole carbon and energy source and grows on a medium containing concentration up to 150 mg/L. A growth medium formulated based on the results of the Plackett-Burman experiment and supplied with 150 mg/L chlorpyrifos recorded 95.12% pesticide decomposition within 48 h. Degradation study of chlorpyrifos by B. subtilis Y242 was examined by gas chromatography–mass spectrometer (GC-MS) and high-performance liquid chromatography (HPLC). These results suggest that B. subtilis Y242 will be potentially useful in the cleanup of contaminated pesticide waste in the environment.

Antifungal and biochemical effects of pseudoguaianolide sesquiterpenes isolated from Ambrosia maritima L.

Five pseudoguaianolide sesquiterpenes, namely neoambrosin, damsinic acid, damsin, ambrosin and hymenin isolated from the aerial parts of Ambrosia maritima, were tested for their antifungal activity against the most economic plant pathogenic fungi Botrytis cinerea and Fusarium oxysporum which cause grey mold and root rot diseases, respectively. In mycelial radial growth inhibition assay, neoambrosin and damsin were the most potent, while hymenin and damsinic acid were the lowest effective among the tested compounds. F. oxysporum was more sensitive for inhibition than B. cinerea. On the other hand, the sesquiterpenes caused significant reduction in spore germination of both fungi at 50, 100 and 200 mg/L compared with the control. Complete inhibition was observed when the spores treated with damsinic acid and ambrosin at 200 mg/L. In vivo activities of polyphenol oxidase, polygalacturonase and pectin-lyase in both fungi that treated with 0.5 and 1 fold of the EC50 value were also carried out in order to investigate the biochemical influences of the tested compounds. The compounds showed significant inhibitory effects on the enzyme activities compared with the control however; the activity was not consistently related to the in vitro inhibitory effects.

Toxicity Assessment of Buprofezin, Lufenuron, and Triflumuron to the Earthworm Aporrectodea caliginosa

Earthworms are particularly important soil macroinvertebrates and are often used in assessing the general impact of pesticide pollution in soil. The present study was conducted in order to investigate the toxicity of three insect growth regulators (IGRs) buprofezin, lufenuron, and triflumuron, at different application rates and exposure times toward mature earthworms Aporrectodea caliginosa. The effects of these pesticides on the growth rate in relation to the activities of acetylcholinesterase (AChE) and glutathione S-transferase (GST) as biochemical indicators were evaluated to elucidate the mechanisms of action. Toxicity studies indicated that lufenuron was the most harmful pesticide to mature earthworms, followed in descending order by buprofezin and triflumuron. A reduction in growth rate in all pesticide-treated worms was dose-dependent over the 28-day exposure period, which was accompanied by a decrease in AChE and GST activities. Relationships between growth rate, AChE, and GST provided strong evidence for the involvement of pesticidal contamination in the biochemical changes in earthworms, which can be used as a bioindicator of soil contamination by pesticides.

Preparation and Characterization of Biopolymers Chitosan/Alginate/Gelatin Gel Spheres Crosslinked by Glutaraldehyde

ABSTRACT Twelve different samples of gel spheres were prepared from the biopolymers chitosan, alginate, and gelatin via polyion complex formation in aqueous solution with crosslinking by glutaraldehyde. Dropwise addition of a chitosan/gelatin solution into a solution containing alginate and glutaraldehyde gave the gel spheres. The effects of different ratios of glutaraldehyde (0.25%, 0.50%, 1.0%, and 2.0%), and gelatin (2.5%, 5.0%, and 10.0%) on the characteristics of the gel spheres were evaluated. An increase in the concentration of the glutaraldehyde led to forming true spheres in rigid form. By scanning electron microscopy (SEM), the gel spheres showed fibrous network propagation along the gel membrane surface. Fourier transform infrared (FT-IR) spectroscopy confirmed the crosslinking of the amino groups by the glutaraldehyde and the presence of crosslinking bonds between the amino groups of chitosan and the carboxyl groups in the alginate molecule. Swelling studies showed that increasing the degree of crosslinking increased the density of the polymer network, which led to a decrease in the degree of swelling. The characteristics of the gel spheres will be useful for immobilization and prolonged release of biologically active substances.

Strawberry Shelf Life, Composition, and Enzymes Activity in Response to Edible Chitosan Coatings

ABSTRACT A series of active biodegradable coatings based on chitosan, gelatin, starch, and sorbitol with or without monoterpenes (geraniol and thymol) were prepared and applied on fresh strawberry fruit as postharvest treatments. The coated fruits were inoculated with fungal spores of Botrytis cinerea and stored for 7 days at 4 °C. Decay incidence, weight loss, anthocyanins, total soluble solids (TSS), total soluble phenolics (TSP), polygalacturonase (PGase), pectin-lyase (PLase), antioxidant activity, guaiacol peroxidase (G-POD), polyphenol oxidase (PPO), and catalase (CAT) were elucidated in the fruits of experimental sets and they were compared with that of controls. The coatings showed a significant effect on the development of quality variables, with the additional effect of geraniol and thymol as a function of the polysaccharide matrix. The coatings inhibited decay incidence, reduced weight loss, delayed changes in the contents of anthocyanin, TSS, and total soluble phenolics; inhibited the increase in G-POD and PPO activities; and retarded the reduction in CAT activity. Compared to the controls, all of the coatings had positive effects on the inhibition of cell wall degrading enzymes (PGase and PLase) and among all the tested coatings, T5, chitosan (1%) + starch (1%) + sorbitol (0.5%) + tween (0.05%) + thymol (0.02%) was the best. This formulation showed also the highest antimicrobial activity and the greatest effect on other physiochemical parameters and it can be suggested to use it as a useful coating agent for extending the shelf-life and maintaining quality of strawberry fruit.

Inhibitory effects on microbial growth of Botrytis cinerea and Erwinia carotovora on potato using of a biopolymer chitosan at different molecular weights

The antimicrobial efficiency of chitosan at different molecular weights (5, 37, 57 and 290 kDa) against Botrytis cinerea and Erwinia carotovora on potato (Solanum tuberosum L.) was investigated. In vitro study showed that chitosan of 37 kDa was the most active against E. carotovora (minimum inhibitory concentration (MIC) = 950 mg/L), whereas 5 kDa chitosan was the most active against B.cinerea. Coating of potato tubers with 100, 250 and 500 mg/L significantly decreased the rate of weight loss and chitosan of 37 kDa showed the best effect. The in vivo antibacterial effect indicated that all treatments (500, 1000 and 2000 mg/L) significantly inhibited the growth of E. carotovora compared with the control. The lowest decay incidence was observed with 37 kDa chitosan. However, the antifungal activity against B. cinerea inoculated of leaves showed no decay incidence at 500 and 1000 mg/L with 57 kDa chitosan after 48 h.

Development and validation of HPLC methods for analysis of chlorantraniliprole insecticide in technical and commercial formulations

ABSTRACT Effective, selective, precise and accurate liquid chromatographic analytical methods for the analysis of a novel chlorantraniliprole insecticide in technical and formulation (coragen, 20% SC) have been optimized and validated. Eight methods were designed based on different mobile phases, temperature and two HPLC columns. The mobile phase consists of two mixtures (acetonitrile:water, 70:30 and methanol:water, 70:30) with 25 or 40ºC. HPLC analysis of chlorantraniliprole was carried out at a wavelength of 260 nm, with a flow rate of 0.8 mL/min. The calibration curves showed a good linear relationship (R2 ˃ 0.99) in the injected quantities ranged from 0.0125 to 1.00 μg. Limit of detection (LOD) was found to be 3.94 to 14.56 ng and from 5.95 to 12.93 ng using the analytical methods I to IV by MicroPack CN-10 and V-VIII by ZORBAX Eclips Plus C18 columns, respectively, based on SDslope values. ZORBAX Eclips Plus C18 column with method VI was the best one (R2 = 1.00 and RSD = 0.30), short retention time (4.936 min), high theoretical plates per column (65457.15) compared to others and LOD = 6.49 ng. The accuracy of the best method was demonstrated by recovery rates of 83.04% to 98.50% for grape samples supplemented with 5, 10 and 50 mg chlorantraniliprole/kg.