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Abstract

Root-knot nematodes (RKNs) are a vital pest that causes significant yield losses and economic damage to potato plants. The use of chemical pesticides to control these nematodes has led to environmental concerns and the development of resistance in the nematode populations. Endophytic fungi offer an eco-friendly alternative to control these pests and produce secondary metabolites that have nematicidal activity against RKNs. The objective of this study is to assess the efficacy of Aspergillus flavus (), an entophyte fungus isolated from Trigonella foenum-graecum seeds, against Meloidogyne incognita in filtered culture broth using GC–MS analysis. Among them, various nematicidal secondary metabolites were produced: Gadoleic acid, Oleic acid di-ethanolamide, Oleic acid, and Palmitic acid. In addition, biochemical compounds such as Gallic acid, Catechin, Protocatechuic acid, Esculatin, Vanillic acid, Pyrocatechol, Coumarine, Cinnamic acid, 4, 3-indol butyl acetic acid and Naphthyl acetic acid by HPLC. The fungus was identified through morphological and molecular analysis, including ITS 1–4 regions of ribosomal DNA. In vitro experiments showed that culture filtrate of A. flavus had a variable effect on reducing the number of egg hatchings and larval mortality, with higher concentrations showing greater efficacy than Abamectin. The fungus inhibited the development and multiplication of M. incognita in potato plants, reducing the number of galls and eggs by 90% and 89%, respectively. A. flavus increased the activity of defense-related enzymes Chitinas, Catalyse, and Peroxidase after 15, 45, and 60 days. Leaching of the concentrated culture significantly reduced the second juveniles’ stage to 97% /250 g soil and decreased the penetration of nematodes into the roots. A. flavus cultural filtrates via soil spraying improved seedling growth and reduced nematode propagation, resulting in systemic resistance to nematode infection. Therefore, A. flavus can be an effective biological control agent for root-knot nematodes in potato plants. This approach provides a sustainable solution for farmers and minimizes the environmental impact.

Abstract

Background/Objectives: Adaptation can reduce or completely eliminate the effectiveness of antibiotics and antiseptics at clinical concentrations. To our knowledge, no studies have examined fungal adaptation to antiseptics. This study aimed to preliminarily investigate the potential for Candida albicans adaptation to eight antiseptics. Methods: The minimal inhibitory concentration (MIC), drug susceptibility, adaptation to antiseptics, and Karpinski Adaptation Index (KAI) of C. albicans strains were assessed. Results: The antiseptics with the most effective MICs activity against C. albicans were octenidine dihydrochloride (OCT), chlorhexidine digluconate (CHX), and polyhexamethylene biguanide (polyhexanide, PHMB). Sodium hypochlorite (NaOCl) and ethacridine lactate (ET) demonstrated moderate activity, while boric acid (BA), povidone–iodine (PVI), and potassium permanganate (KMnO4) showed the weakest activity. The MIC values for NaOCl and KMnO4 were close to or equal to the clinical concentrations used in commercial products. The studied strains were susceptible to econazole, miconazole, and voriconazole. Resistance to other drugs occurred in 10–30% of the strains. Antifungal resistance remained unchanged after antiseptic adaptation testing. The lowest KAI values, indicating very low resistance risk, were observed for CHX, OCT, and PHMB. PVI and BA presented a low risk, ET a moderate risk. KMnO4 and NaOCl had the highest KAI values, indicating high and very high resistance risk in Candida yeasts. Conclusions: C. albicans strains can adapt to antiseptics to varying extents. For most antiseptics, adaptation does not significantly affect their clinical efficacy. However, due to adaptation, NaOCl and KMnO4 may become ineffective against C. albicans strains even at clinical concentrations.

2.3. Adaptation of Candida albicans Strains to Antiseptics

The studies demonstrated that C. albicans yeasts undergo adaptation when exposed to increasing concentrations of antiseptics. The strains showed weak adaptation to OCT, CHX, PVI, and ET, managing to grow at concentrations several times higher than their respective MIC values. For NaOCl and KMnO₄, adaptation levels were also low but were close to or equal to the initial commercial concentration. This finding suggests that the development of adaptation may lead to the ineffectiveness of commercial products containing NaOCl and KMnO4 against C. albicans. The highest increase in adaptation relative to MIC was observed for BA and PHMB; however, these values were still much lower than the commercial concentrations.

2.3. Adaptation of Candida albicans Strains to Antiseptics

Based on the Karpinski Adaptation Index, the lowest KAI values were observed for CHX, OCT, and PHMB, indicating a very low risk of clinical resistance development in the studied C. albicans strains. PVI and BA had a low risk, while ET posed a moderate risk for clinical resistance development. The highest KAI values were observed for KMnO₄ and NaOCl, indicating a high and very high risk, respectively, of clinical resistance development in Candida yeasts. The results of C. albicans adaptation to antiseptics are provided in . Detailed raw data on the C. albicans adaptation are presented in the .

4.1. Antiseptics

Eight antiseptics were examined in the study: octenidine dihydrochloride (OCT; Schülke & Mayr GmbH, Norderstedt, Germany), polyhexamethylene biguanide (PHMB, Cosmocil PG; Arxada AG, Basel, Switzerland), sodium hypochlorite (NaOCl; Cerkamed, Stalowa Wola, Poland), potassium permanganate (KMnO4; Hasco-Lek S.A., Wrocław, Poland), chlorhexidine digluconate (CHX; Sigma-Aldrich, Poznań, Poland), povidone–iodine (PVI, poly(vinylpyrrolidone)–iodine complex; Sigma-Aldrich, Poznań, Poland), boric acid (BA, Borasol; Herbapol, Poznań, Poland), and ethacridine lactate (ET, Rivanol; Herbapol, Poznań, Poland). The initial concentrations of active substances matched the clinical concentrations found in commercial products used for wound and oral antiseptics: 0.05% (500 µg/mL) OCT, 0.1% (1000 µg/mL) PHMB, 0.01% (100 µg/mL) NaOCl, 1% (10 mg/mL) KMnO4, 0.1% (1000 µg/mL) CHX, 7.5% (75 mg/mL) PVI, 3% (30 mg/mL) BA, and 0.1% (1000 µg/mL) ET.

Abstract

Gene therapy offers potentially transformative strategies for major human diseases. However, one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissue. Here, we report a novel virus-like nanoparticle, the bioorthgonal engineered virus-like recombinant biosome (reBiosome), for efficient gene therapies of cancer and inflammatory diseases. The mutant virus-like biosome (mBiosome) is first prepared by site-specific codon mutation for displaying 4-azido-L-phenylalanine on vesicular stomatitis virus glycoprotein of eBiosome at a rational site, and the reBiosome is then prepared by clicking weak acid-responsive hydrophilic polymer onto the mBiosome via bioorthogonal chemistry. The results show that the reBiosome exhibits reduced virus-like immunogenicity, prolonged blood circulation time and enhanced gene delivery efficiency to weakly acidic foci (like tumor and arthritic tissue). Furthermore, reBiosome demonstrates robust therapeutic efficacy in breast cancer and arthritis by delivering gene editing and silencing systems, respectively. In conclusion, this study develops a universal, safe and efficient platform for gene therapies for cancer and inflammatory diseases.

Site-Specific Codon Mutated Virus-Like mBiosome and Its Gene Transfection Efficiency

To prepare the mutated VSV-G enveloped biosome packaging the GFP mRNA (mBiosomeGFP), a series of steps were undertaken. Firstly, HEK 293T cells were co-transfected with the mutated pCMV-VSV-G plasmid along with other three plasmids, pIRE4-Azi, pCMV-capsid10-flag (encoding the capsid10 protein), and pCMV-cargoGFP (encoding the GFP mRNA). This transfection process was carried out in the presence of 1 mM Azi, as depicted in Fig. c. After the concentration of mBiosomes, the membrane proteins were extracted and analyzed by Western blot assay. The results clearly demonstrated a successful incorporation of mutated VSV-G proteins into the envelope of mBiosomes (Fig. d). In order to gain further insights, the morphologies, particle sizes and Zeta potentials of the mBiosomes were examined using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The TEM images showed that eBiosome had a spherical shape and was encapsulated with spike proteins, closely resembling general enveloped virus. Similarly, three different types of mBiosomes displayed similar morphological characteristics to the eBiosome (Fig. e). Moreover, the particle sizes and Zeta potential values of the three different types of mBiosomesGFP were evaluated and compared to the eBiosomeGFP. The results obtained from DLS analysis demonstrated that all three types of mBiosomesGFP shared comparable particle sizes and Zeta potential values with the eBiosomeGFP (Figs. f and S3).

Expression of plant EFE-like proteins causes guanidine production in E. coli

The detection of free guanidine indicated that 2-ODD-C23 might be homoarginine-6-hydroxylases, as 6-hydroxyhomoarginine is expected to decay spontaneously to yield guanidine and AASA. The latter is in spontaneous equilibrium with P6C, which can be converted to pipecolate by P5CR (proC in E. coli, ). Indeed, we detected pipecolate in the E. coli cultures in quantities proportional to the amount of guanidine (). Only in the cells expressing Din11s, the proportion of pipecolate was lower, suggesting that Din11s used both arginine and homoarginine as substrates. In contrast to guanidine, pipecolate was predominantly retained inside the cells (). To analyze if the plant enzymes can convert 2-oxoglutarate to ethylene, we compared bacterial cultures expressing either PsEFE or one of the Arabidopsis enzymes (). By GC-FID analysis of the headspace of bacterial cultures expressing EFE, Din11s, At3g49630, or At3g50210 and incubated with 1 mM arginine or homoarginine for 18 hr in air-tight tubes, we observed the formation of up to 0.18% (v/v) ethylene by cultures expressing EFE. In contrast, none of the plant enzymes produced even traces of ethylene [detection limit 0.0002% (v/v)]. As expected, the cultures expressing EFE or Din11s contained guanidine, irrespective of the supplement. The cultures expressing At3g49630 or At3g50210 only contained guanidine, when they were fed with homoarginine.

Introduction

Only two enzymes are known to release guanidine from arginine in side reactions. The Ethylene Forming Enzyme (EFE) from Pseudomonas savastanoi converts 2-oxoglutarate (2-OG) into ethylene and this activity depends on structural changes induced by binding of arginine to the active site (; ). In a second reaction with roughly 50% turnover rate, EFE also catalyzes the hydroxylation of arginine at the C5 atom, yielding unstable 5-hydroxyarginine, which spontaneously decays to glutamate-5-semialdehyde (GSA) and guanidine. The arginine-4,5-desaturase NapI predominantly catalyzes desaturation of the C4-C5 bond but also produces a small proportion unstable 5-hydroxyarginine (). Both enzymes belong to the class of Fe2+ and 2-OG-dependent dioxygenases (2-ODD), which use molecular oxygen to catalyze a broad range of reactions typically involving an FeIV-oxo-intermediate formed after the initial conversion of 2-OG to succinate (; ; ). Other enzymes from this family, like VioC or OrfP, catalyze the hydroxylation of arginine at C3 or C3 and C4, respectively, without initiating guanidine release (; ). A third guanidine-producing enzyme, the guanylurea hydrolase GuuH from Pseudomonas mendocina strain GU, is from a different protein family and may have evolved recently to utilize guanylurea generated by metformin degradation ().