Summary: 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.

Proline metabolism influences the metabolic and/or signaling pathway in regulating plant stress responses. This study aimed to characterize the physiological significance of glutamate (Glu)-mediated proline metabolism in the drought stress responses, focusing on the hormonal regulatory pathway. The responses of cytosolic Ca²⁺ signaling, proline metabolism, and redox components to the exogenous application of Glu in well-watered or drought-stressed plants were interpreted in relation to endogenous hormone status and their signaling genes. Drought-enhanced level of abscisic acid (ABA) was concomitant with the accumulation of ROS and proline, as well as loss of reducing potential, which was assessed by measuring NAD(P)H/NAD(P)⁺ and GSH/GSSG ratios. Glu application to drought-stressed plants increased both salicylic acid (SA) and cytosolic Ca²⁺ levels, with the highest expression of calcium-dependent protein kinase (CPK5) and salicylic acid synthesis-related ICS1. The SA-enhanced CPK5 expression was closely associated with further enhancement of proline synthesis-related genes (P5CS1, P5CS2, and P5CR) expression and a reset of reducing potential with enhanced expression of redox regulating genes (TRXh5 and GRXC9) in a SA-mediated NPR1- and/or PR1-dependent manner. These results clearly indicate that Glu-activated interplay between SA- and CPK5-signaling as well as Glu-enhanced proline synthesis are crucial in the amelioration of drought stress in Brassica napus.

Botrytis cinerea is one of the most critical agro-economic phytopathogens and has been reported to cause gray mold disease in more than 1000 plant species. Meanwhile, small interfering RNA (siRNA), which induce RNA interference (RNAi), are involved in both host immunity and pathogen virulence. B. cinerea has been reported to use both siRNA effectors and host RNAi machinery to facilitate the progression of gray mold in host species. Accordingly, RNAi-based biofungicides that use double-stranded RNA (dsRNA) to target essential fungal genes are considered an emerging approach for controlling devastating gray mold diseases. Furthermore, spray-induced gene silencing (SIGS), in which the foliar application of dsRNA is used to silence the pathogen virulence genes, holds great potential as an alternative to host-induced gene silencing (HIGS). Recently, SIGS approaches have attracted research interest, owing to their ability to mitigate both pre- and post-harvest B. cinerea infections. The RNAi-mediated regulation of host immunity and susceptibility in B. cinerea–host interactions are summarized in this review, along with the limitations of the current knowledge of RNAi-based biofungicides, especially regarding SIGS approaches for controlling gray mold diseases under field conditions.

This study aimed to directly elucidate cultivar variation in disease susceptibility and disease responses in relation to hormonal status in the interaction of Brassica napus cultivars and Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot disease. Fully expanded leaves of six B. napus cultivars (cvs. Capitol, Youngsan, Saturnin, Colosse, Tamra, and Mosa) were inoculated with Xcc. At 14 days post-inoculation with Xcc, cultivar variation in susceptibility or resistance was interpreted with defense responses as estimated by redox status, defensive metabolites, and expression of phenylpropanoid synthesis-related genes in relation to endogenous hormonal status. Disease susceptibility of six cultivars was distinguished by necrotic lesions in the Xcc-inoculated leaves and characterized concurrently based on the higher increase in reactive oxygen species and lipid peroxidation. Among these cultivars, as the susceptibility was higher, the ratios of abscisic acid (ABA)/jasmonic acid (JA) and salicylic acid (SA)/JA tended to increase with enhanced expression of SA signaling regulatory gene NPR1 and transcriptional factor TGA1 and antagonistic suppression of JA-regulated gene PDF 1.2. In the resistant cultivar (cv. Capitol), accumulation of defensive metabolites with enhanced expression of genes involved in flavonoids (chalcone synthase), proanthocyanidins (anthocyanidin reductase), and hydroxycinnamic acids (ferulate-5-hydroxylase) biosynthesis and higher redox status were observed, whereas the opposite results were obtained for susceptible cultivars (cvs. Mosa and Tamra). These results clearly indicate that cultivar variation in susceptibility to infection by Xcc was determined by enhanced alteration of the SA/JA ratio, as a negative regulator of redox status and phenylpropanoid synthesis in the Brasica napus–Xcc pathosystem.

Spring frosts exacerbated by global climate change have become a constant threat to temperate fruit production. Delaying the bloom date by plant growth regulators (PGRs) has been proposed as a practical frost avoidance strategy. Ethephon is an ethylene-releasing PGR found to delay bloom in several fruit species, yet its use is often coupled with harmful effects, limiting its applicability in commercial tree fruit production. Little information is available regarding the mechanisms by which ethephon influences blooming and bud dormancy. This study investigated the effects of fall-applied ethephon on bud phenology, cold hardiness, and hormonal balance throughout the bud dormancy cycle in peach. Our findings concluded that ethephon could alter several significant aspects of peach bud physiology, including accelerated leaf fall, extended chilling accumulation period, increased heat requirements, improved cold hardiness, and delayed bloom date. Ethephon effects on these traits were primarily dependent on its concentration and application timing, with a high concentration (500 ppm) and an early application timing (10% leaf fall) being the most effective. Endogenous ethylene levels were induced significantly in the buds when ethephon was applied at 10% versus 90% leaf fall, indicating that leaves are essential for ethephon uptake. The hormonal analysis of buds at regular intervals of chilling hours (CH) and growing degree hours (GDH) also indicated that ethephon might exert its effects through an abscisic acid (ABA)-independent way in dormant buds. Instead, our data signifies the role of jasmonic acid (JA) in mediating budburst and bloom in peach, which also appears to be influenced by ethephon treatment. Overall, this research presents a new perspective in interpreting horticultural traits in the light of biochemical and molecular data and sheds light on the potential role of JA in bud dormancy, which deserves further attention in future studies that aim at mitigating spring frosts.

Understanding the biochemical mechanisms underlying bud dormancy and bloom time regulation in deciduous woody perennials is critical for devising effective strategies to protect these species from spring frost damage. This study investigated the accumulation profiles of carbohydrates, ROS and antioxidants during dormancy in ‘Cripps Pink’ and ‘Honeycrisp’, two apple cultivars representing the early and late bloom cultivars, respectively. Our data showed that starch levels generally declined during dormancy, whereas soluble sugars increased. However, the present study did not record significant alternations in the carbohydrate accumulation profiles between the two cultivars that could account for the differences in their bloom dates. On the other hand, H₂O₂ accumulation patterns revealed an apparent correlation with the dormancy stage and bloom dates in both cultivars; peaking early in the early-blooming cultivar, sustaining high levels for a longer time in the late-blooming cultivars, and fading by the time of bud burst in both cultivars. Also, the redox balance during dormancy appeared to be maintained mainly by catalase and, to a lesser extent, by glutathione (GSH). Overall, the present study concludes that differences in ROS and the bud redox balance could, at least partially, explain the differences in dormancy duration and bloom date among apple cultivars.