Synthesis and antifungal activities of hydrophilic cationic polymers against Rhizoctonia solani

Abstract

A series of linear hydrophilic cationic polymers with different charge density and molecular weights were synthesized by one-step polymerization process. The effect of the hydrophobicity and molecular weights on the antifungal activity against Rhizoctonia solani (R. solani) and Fusarium oxysporum f. sp. cubense race 4 (Foc4) was assessed. The biotoxicity of the cationic polymers were evaluated based on their median lethal concentration (LC₅₀) for zebrafish and silkworm and median lethal dose (LD₅₀) for Kunming mice. The results indicated that the balance between antifungal activity and biotoxicity could be well tuned by controlling the hydrophobic-hydrophilic balance. The minimum inhibitory concentration (MIC) of PEPB10 and PEPB25 against R. solani were 160 μg/mL and 80 μg/mL, respectively. And the LD₅₀ for Kunming mice of PEPB10 and PEPB25 were more than 5000 mg/kg, which mean that PEPB10 and PEPB25 with high hydrophilicity show low toxicity and better selectivity for R. solani. The cationic polymers can kill the R. solani by damaging their membranes and exchanging the Ca²⁺ or/and Mg²⁺ cations of their membranes or cell wall. These results help to understand the antifungal mechanism of low-toxic polymeric quaternary ammonium salts and highlight their potential application as highly selective fungicidal agents for controlling plant diseases.

Introduction

Fungal disease is the main risk to agricultural development throughout the world. For example, rice sheath blight (Rhizoctonia solani) is a devastating disease in intense and high-input rice production systems, which account for most of the global consumption of rice fungicide (Wu et al., 2013). And the rice sheath blight was controlled mainly through chemical control at present. The heavy use of existing pesticides may lead to resistance for the phytopathogenic fungi. In the last twenty years, the R. solani that had produced drug resistance to existing pesticides were found in various regions of China (Cheng, 2014). Cationic polymers possess potent broad-spectrum antimicrobial activity (Xie et al., 2011, Rahman et al., 2018, Venkataraman et al., 2019, Peng et al., 2019) and do not elicit antimicrobial resistance (Choi et al., 2017, Lou et al., 2018). The cationic polymers have potential application as fungicidal agents for controlling plant diseases (see Scheme 1).

Toxicity is a significant factor to affect the application of pesticides. The heavy use of existing pesticides may lead to risk for environmental ecosystem especially for aquatic organisms. According to reports, pesticides have been detected and threatened aquatic ecosystems around the world (Castillo et al., 2000, Pandit et al., 2001, Sarkar et al., 2008). Unlike low molecular weight antimicrobial agents, the antimicrobial polymers show low toxicity, better efficacy and selectivity (Kenawy et al., 2007, Chen et al., 2000, Koromilas et al., 2016). In addition, the hydrophobic moiety of cationic polymers is a key factor in balancing antimicrobial activities and toxicity (Engler et al., 2012). Larger hydrophobic groups will have stronger interactions with the inner hydrophobic core of cell membranes, which lead to the increase in antimicrobial activity and hemolytic activity and decrease in selectivity (Ilker et al., 2004, van’t Hof et al., 2001). Oda and co-workers discovered that the block copolymers are much less hemolytic compared to the highly hemolytic random copolymers. The block copolymers could form intramolecular aggregates with a hydrophobic core wrapped by the cationic segment in water, which can reduce the interactions between hydrophobic groups and cell membranes (Oda et al., 2011). Additionally, appropriate reduction of hydrophobicity is conducive to reducing the biotoxicity of cationic polymers. Locock and co-workers found that Guanidine copolymers of low to moderate molecular weight and hydrophobicity had high antimicrobial activity with low toxicity (Locock et al., 2013). Strassburg and co-workers synthesized a series of linear hydrophilic quaternized antimicrobial polymers which are not hemocytotoxic and found that the linear hydrophilic cationic polymers proposed that their bacteria-killing mechanism might not be based on membrane disruption (Strassburg et al., 2015).

The antimicrobial mechanism of cation polymers is complicated and has been continuously explored. It is considered that the antimicrobial activity of amphiphilic cationic polymers was provided by both hydrophilic cation and hydrophobic moieties (Majumdar et al., 2009). The hydrophilic cation bind to the negatively charged phospholipid of bacterial membrane and the hydrophobic moieties interact with the inner hydrophobic core of the bacterial membrane leading to a disruption in integrity and subsequent cell death (Sovadinova et al., 2011, Paslay et al., 2012). In addition, the cation of antimicrobial agent can displace by exchange the Ca²⁺ or/and Mg²⁺ cations of the outer membrane and cell wall, bring about a disruption of the membrane and cell wall (Gilbert and Moore, 2005, Lenoir et al., 2006, Thomas and Rice, 2014, Huang et al., 2008, Qian et al., 2018). According to our previous studies, the quaternary ammonium salt have several targets in fungal cells, including the disruption of cellular structures, such as the cell wall and plasma membrane; the induction of lipid peroxidation; mitochondrial dysfunction and interference with genomic DNA (Huang et al., 2017, Dong et al., 2018).

In our previous studies, a series of acrylate polymers containing quaternary ammonium salts (PDMAEMA-BC) which have a hydrophobic backbone with different molecular weights were synthesized, and the optimal antifungal activities toward R. solani mycelium were achieved when the molecular weights of QPDMAEMA were lower than 5 kDa (Zhong et al., 2017). In addition, polydimethylsiloxane-polymethacrylate block copolymers containing quaternary ammonium salts (PDMS-b-QPDMAEMA) were synthesized, and the moderately hydrophobic PDMS blocks help stabilize the attachment of the amphiphilic quaternary ammonium salt and then help it penetrate the R. solani sclerotia and thus effectively inhibit the germination of the sclerotia (Lin et al., 2018). In addition, the results of toxicity tests showed that the cationic polymers which we synthesized were safe for mammals and insects. And the relationship between structural characteristics and the toxicity to aquatic organisms need further studies. The charge density and hydrophobic-hydrophilic balance have important effects on the antifungal activities and biological toxicity of cationic polymers. Proper hydrophobicity is the key to obtain high antifungal activities of cationic polymers. However in order to obtain low biological toxicity and high selectivity, it is an effective way to improve the charge density and hydrophilicity of the cationic polymers. We designed a series of linear hydrophilic cationic polymers. Unlike amphiphilic cationic polymers, linear hydrophilic cationic polymers are hydrophilic and have high charge density. By tuning the hydrophilicity of the main chain, the hydrophilic cationic polymers might obtain the balance of low toxicity, high selectivity and high antifungal activities. In this study, we synthesized a series of hydrophilic cationic polymers with different charge density and molecular weights by one-step polymerization process. And the antifungal activities against phytopathogenic fungi (R. solani and Foc4), antifungal mechanism and toxicity to aquatic organisms (zebrafish), insect (silkworm) and mammal (Kunming mice) were systematically assessed.