In vitro assessment of the antimicrobial efficacy of chitosan nanoparticles against major fish pathogens and their cytotoxicity to fish cell lines.

Nanotechnology is an emerging avenue employed in disease prevention and treatment. This study evaluated the antimicrobial efficacy of chitosan nanoparticles (CSNPs) against major bacterial and oomycete fish pathogens in comparison with chitosan suspension. Initially, the minimum inhibitory concentrations (MIC, MIC90 ) were determined and the per cent inhibition of bacterial growth was calculated. Subsequently, the minimum bactericidal concentrations (MBCs) were determined. The time-dependent disruptions of CSNP-treated pathogens were observed via transmission electron microscopy (TEM), and the effect of CSNPs on the viability of two fish cell lines was assessed. No antimicrobial effect was observed with chitosan, while CSNPs (105 nm) exhibited a dose-dependent and species-specific antimicrobial properties. They were bactericidal against seven bacterial isolates recording MBC values from 1 to 7 mg/ml, bacteriostatic against four further isolates recording MIC values from 0.125 to 5 mg/ml and fungistatic against oomycetes recording MIC90 values of 3 and 4 mg/ml. TEM micrographs showed the attachment of CSNPs to the pathogenic cell membranes disrupting their integrity. No significant cytotoxicity was observed using 1 mg/ml CSNPs, while low dose-dependent cytotoxicity was elicited by the higher doses. Therefore, it is anticipated that CSNPs are able to compete and reduce using antibiotics in aquaculture.

Design, Synthesis, and Structure-Activity Relationship of Economical Triazole Sulfonamide Aryl Derivatives with High Fungicidal Activity.

Plant fungal diseases have caused great decreases in crop quality and yield. As one of the considerable agricultural diseases, cucumber downy mildew (CDM) caused by pseudoperonospora cubensis seriously influences the production of cucumber. Amisulbrom is a commercial agricultural fungicide developed by Nissan Chemical, Ltd., for the control of oomycetes diseases that is highly effective against CDM. However, the synthesis of amisulbrom has a high cost because of the introduction of the bromoindole ring. In addition, the continuous use of amisulbrom might increase the risk of resistance development. Hence, there is an imperative to develop active fungicides with new scaffolds but low cost against CDM. In this study, a series of 1,2,4-triazole-1,3-disulfonamide derivatives were designed, synthesized, and screened. Compound 1j showed a comparable fungicidal activity with amisulbrom, but it was low cost and ecofriendly. It has the potential to be developed as a new fungicide candidate against CDM. Further investigations of structure-activity relationship exhibited the structural requirements of 1,2,4-triazole-1,3-disulfonamide and appropriate modification in N-alkyl benzylamine groups with high fungicidal activity. This research will provide powerful guidance for the design of highly active lead compounds with a novel skeleton and low cost.

Soft selective sweeps in fungicide resistance evolution: recurrent mutations without fitness costs in grapevine downy mildew.

Adaptation produces hard or soft selective sweeps depending on the supply of adaptive genetic polymorphism. The evolution of pesticide resistance in parasites is a striking example of rapid adaptation that can shed light on selection processes. Plasmopara viticola, which causes grapevine downy mildew, forms large populations, in which resistance has rapidly evolved due to excessive fungicide use. We investigated the pathways by which fungicide resistance has evolved in this plant pathogen, to determine whether hard or soft selective sweeps were involved. An analysis of nucleotide polymorphism in 108 field isolates from the Bordeaux region revealed recurrent mutations of cytb and CesA3 conferring resistance to quinone outside inhibiting (QoI) and carboxylic acid amide (CAA) fungicides, respectively. Higher levels of genetic differentiation were observed for nucleotide positions involved in resistance than for neutral microsatellites, consistent with local adaptation of the pathogen to fungicide treatments. No hitchhiking was found between selected sites and neighbouring polymorphisms in cytb and CesA3, confirming multiple origins of resistance alleles. We assessed resistance costs, by evaluating the fitness of the 108 isolates through measurements of multiple quantitative pathogenicity traits under controlled conditions. No significant differences were found between sensitive and resistant isolates, suggesting that fitness costs may be absent or negligible. Our results indicate that the rapid evolution of fungicide resistance in P. viticola has involved a soft sweep.

Producing cell-free culture broth of rhamnolipids as a cost-effective fungicide against plant pathogens.

Biosurfactants of rhamnolipids have been enthusiastically investigated for substitutes of synthetic agrochemicals against plant pathogens. However, all such studies have been conducted on rhamnolipids with high purity which have limitations due to high costs. This paper focused on the applicability of rhamnolipid-containing cell-free culture broth. It was found that rhamnolipids in cell-free culture broth of Pseudomonas aeruginosa ZJU211 were largely composed of di-rhamnolipid and mono-rhamnolipid with the ratio varying with culture time. After 96 h of fermentation, the mass ratio of di-rhamnolipid over mono-rhamnolipid increased to 2.6:1. Crude rhamnolipids in a form of cell-free culture broth showed high antifungal activity against colony growth and biomass accumulation of seven plant pathogens comprising two Oomycetes, three Ascomycota and two Mucor spp. fungi, among which three plant pathogens were firstly reported in this paper showing inhibition to rhamnolipids. Particularly, rhamnolipids showed potent activity against two Oomycetes that acquire resistance to commercial compound of metalaxyal. Furthermore, di-rhamnolipid was elucidated to dominate the antifungal activity of crude rhamnolipids by in vitro studies. At last, the efficacy and safety of cell-free culture broth was preliminarily illustrated on plants in vivo. So cell-free culture broth as a crude rhamnolipid product could be served as a potential cost-effective and environmental-friendly fungicide in agriculture.

Natural products alone or with copper vs. grape downy mildew: efficacy, costs, Cu impact.

In organic farming copper is one of the few fungicides that can be used and the only effective against downy mildew. Due to the problems of environmental impact associated with the use of this heavy metal, the European Union decided to restrict its use (Annex II Regulation EC n. 889/2008). In order to assess the possibility of replacing the copper or reduce the quantities used, we tested natural products and low rate copper formulations in field trials. The anti-downy mildew activity of these formulations has been studied in an organic vineyard. During the two years of activity we also estimated the economic cost of use of the products to assess their possible use in agricultural practice.

Assessment of QoI resistance in Plasmopara viticola oospores.

QoI fungicides, inhibitors of mitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc(1) enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Berl. & De Toni. Numerous treatments per year with QoI fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. QoI resistance was not observed in one vineyard never treated with QoI fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of QoI-resistant strains was lower, compared with using solely QoI. In vineyards where QoI treatments have been stopped, a decrease in resistance was generally observed.

A Saprolegnia parasitica challenge system for rainbow trout: assessment of Pyceze as an anti-fungal agent for both fish and ova.

A reproducible Saprolegnia parasitica spore delivery system was developed and demonstrated to be effective in providing a sustained spore challenge for up to 10 d. Treatment of rainbow trout with slow-release intraperitoneal implants containing cortisol resulted in chronically elevated blood cortisol levels and rendered the fish susceptible to infection by S. parasitica when exposed to the spore challenge. Sham-implanted fish were not susceptible to infection. Bronopol (2-bromo-2-nitro-propane-1,3-diol), formulated as Pyceze, was effective in protecting predisposed fish from infection by S. parasitica when administered as a daily bath/flush treatment at concentrations of 15 mg l-1 and greater. Pyceze was also demonstrated to protect fertilised rainbow trout ova from S. parasitica challenge when administered as a daily bath/flush treatment at concentrations of between 30 and 100 mg l-1. Pyceze appears to qualify as a safe and effective replacement for malachite green and formalin in the prevention of fungal infections in the aquaculture environment.