Minicell-based fungal RNAi delivery for sustainable crop protection.

Spray-induced gene silencing (SIGS) using topical dsRNA applications has risen as a promising, target-specific, and environmentally friendly disease management strategy against phytopathogenic fungi. However, dsRNA stability, efficacy, and scalability are still the main constraints facing SIGS broader application. Here we show that Escherichia coli-derived anucleated minicells can be utilized as a cost-effective, scalable platform for dsRNA production and encapsulation. We demonstrated that minicell-encapsulated dsRNA (ME-dsRNA) was shielded from RNase degradation and stabilized on strawberry surfaces, allowing dsRNA persistence in field-like conditions. ME-dsRNAs targeting chitin synthase class III (Chs3a, Chs3b) and DICER-like proteins (DCL1 and DCL2) genes of Botryotinia fuckeliana selectively knocked-down the target genes and led to significant fungal growth inhibition in vitro. We also observed a compensatory relationship between DCL1 and DCL2 gene transcripts, where the silencing of one gene upregulated the expression of the other. Contrary to naked-dsRNAs, ME-dsRNAs halted disease progression in strawberries for 12 days under greenhouse conditions. These results elucidate the potential of ME-dsRNAs to enable the commercial application of RNAi-based, species-specific biocontrols comparable in efficacy to conventional synthetics. ME-dsRNAs offer a platform that can readily be translated to large-scale production and deployed in open-field applications to control grey mould in strawberries.

Lenzimycins A and B, Metabolites With Antibacterial Properties From Brevibacillus sp. Associated With the Dung Beetle Onthophagus lenzii.

Symbiotic microorganisms associated with insects can produce a wide array of metabolic products, which provide an opportunity for the discovery of useful natural products. Selective isolation of bacterial strains associated with the dung beetle, Onthophagus lenzii, identified two strains, of which the antibiotic-producing Brevibacillus sp. PTH23 inhibited the growth of Bacillus sp. CCARM 9248, which is most closely related to the well-known entomopathogen, Bacillus thuringiensis. A comprehensive chemical investigation based on antibiotic activity discovered two new antibiotics, named lenzimycins A and B (1-2), which inhibited growth of Bacillus sp. CCARM 9248. The 1H and 13C NMR, MS, MS/MS, and IR analyses elucidated the structures of 1 and 2, which comprised a novel combination of fatty acid (12-methyltetradecanoic acid), glycerol, sulfate, and N-methyl ethanolamine. Furthermore, the acid hydrolysis of 1 revealed the absolute configuration of 12-methyltetradecanoic acid as 12S by comparing its optical rotation value with authentic (R)- and (S)-12-methyltetradecanoic acid. In addition to inhibition of Bacillus sp. CCARM 9248, lenzimycins A and B were found to inhibit the growth of some human pathogenic bacteria, including Enterococcus faecium and certain strains of Enterococcus faecalis. Furthermore, the present study elucidated that lenzimycins A and B activated a reporter system designed to detect the bacterial cell envelope stress, thereby indicating an activity against the integrity of the bacterial cell wall.