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We developed a formulation of long double-stranded RNA (dsRNA) using interpolyelectrolyte complexes (IPECs) composed of the biopolymers chitosan and alginate, in order to protect the dsRNA from biotic and abiotic factors. Our primary objectives were to enhance stability of dsRNA against environmental nucleases and, secondarily, to mitigate the negative charge of the dsRNA, which may promote foliar uptake. Our approach relies on submicron particles with adjustable surface charge being either positive or negative. Following this approach, we obtained a high encapsulation efficiency of 94 %. Subsequently, we investigated the influence of the charge ratio and total polymer content on the size, size distribution and ζ-potential of the IPECs. We discovered that formulating at low polymer concentrations ≤0.05 g/L with charge ratios of ≤0.9 (+/ ) and ≥ 1.25 (+/ ), respectively, produced <100 nm particles. Furthermore, the IPEC formulation protected dsRNA from enzymatic degradation by RNase III and micrococcal nuclease. In addition, we observed outstanding protection of formulated dsRNA from heat degradation. Experiments on Nicotiana benthamiana plants showed that formulated dsRNA offered protection against tobacco mosaic virus. In essence, this formulation demonstrates versatility for the production of IPECs with customizable size, surface charge, and nucleic acid content.
In this study, a novel approach was developed to increase the intracellular trehalose concentration in M. brunneum blastospores by uptake from the medium, improving their desiccation tolerance. Briefly, placing the blastospores in a hypotonic environment induced solute excretion, allowing significantly increased trehalose uptake during subsequent trehalose treatment. Conductivity changes and GC–MS analysis revealed that glycerol and lactate excretion is triggered in a hypotonic NaCl solution. After hypotonic + trehalose treatment, intracellular glycerol, lactate, and glucose levels increased, while other desiccation‑protective solutes remained unaffected. A fluorescence‑based single‑cell analysis of membrane stress and death during osmotic desiccation and rehydration showed that membrane stress and cell death were strongly amplified upon rehydration. Trehalose treatment reduced this amplification, while hypotonic + trehalose treatment minimised cell death and prevented a stress response during rehydration, though it increased membrane stress during desiccation. Newly formed germ tubes were identified as critical points of membrane failure during desiccation. Ultimately, the drying survival of blastospores was significantly improved by the hypotonic + trehalose treatment compared to untreated or trehalose‑only‑treated blastospores. In summary, this technique successfully enhanced blastospore desiccation tolerance and could be adapted to load cells with other solutes.
In vertebrates and plants, dsRNA plays crucial roles as PAMP and as a mediator of RNAi. How higher fungi respond to dsRNA is not known. We demonstrate that Magnaporthe oryzae (Mo), a globally significant crop pathogen, internalizes dsRNA across a broad size range of 21 to about 3000 bp. Incubation of fungal conidia with 10 ng/μL dsRNA, regardless of size or sequence, induced aberrant germ tube elongation, revealing a strong sequence-unspecific effect of dsRNA in this fungus. Accordingly, the synthetic dsRNA analogue poly(I:C) and dsRNA of various sizes and sequences elicited canonical fungal stress pathways, including nuclear accumulation of the stress marker mitogen-activated protein kinase Hog1p and production of ROS. Leaf application of dsRNA to the cereal model species Brachypodium distachyon suppressed the progression of leaf blast disease. Notably, the sequence-unspecific effect of dsRNA depends on higher doses, while pure sequence-specific effects were observed at low concentrations of dsRNA ( < 0.03 ng/μL). The protective effects of dsRNA were further enhanced by maintaining a gap of at least seven days between dsRNA application and inoculation, and by stabilising the dsRNA in alginate-chitosan nanoparticles. Overall, our study opens up additional possibilities for the development and use of dsRNA pesticides in agriculture.
Seed treatment is a powerful technique for adding beneficial ingredients to plants during the seed preparation process. Biopolymers as drying agents and delivery systems in seed treatments were investigated for their biocompatibility with blastospores of the nematophagous fungus Pochonia chlamydosporia. To produce a novel seed treatment for the cover crop Phacelia tanacetifolia, xanthan gum TG and gellan gum were the most promising biopolymers in combination with potato starch and bentonite. The seed treatment process as well as the drying process were specially designed to be scalable, which make it suitable for applying the developed seed treatment in agriculture. Application of gellan gum in seed treatments led to 6.3% ± 1.6% of vital blastospores per seed compared to 3.8% ± 0.3% of vital blastospores when applying xanthan gum. Storage tests for seed treatments with 0.5% gellan gum indicated a higher stability at 4 °C compared to storage at 21 °C. After 42 days of storage at 4 °C, 54.1% ± 15.1% of the applied blastospores were viable compared to 0.3% ± 0.8% at 21 °C. This novel seed treatment application with P. chlamydosporia blastospores includes the seed treatment procedure, drying process, and storage tests and can easily be upscaled for application in agriculture.

