A hallmark of antiviral RNA interference (RNAi) is the production of viral small interfering RNA (vsiRNA). Profiling of vsiRNAs indicates that certain regions of viral RNA genome or transcribed viral RNA, dubbed vsiRNA hotspots, are more prone to RNAi-mediated cleavage for vsiRNA biogenesis. However, the biological relevance of hotspot vsiRNAs to the host innate defence against pathogens remains to be elucidated. Here, we show that direct targeting a hotspot by a synthetic vsiRNA confers host resistance to virus infection. Using Northern blotting and RNAseq, we obtained a profile of vsiRNAs of the African cassava mosaic virus (ACMV), a single-stranded DNA virus. Sense and anti-sense strands of small RNAs corresponding to a hotspot and a coldspot vsiRNA were synthesised. Co-inoculation of Nicotiana benthamiana with the double-stranded hotspot siRNA protected plants from ACMV infection, where viral DNA replication and accumulation of viral mRNA were undetectable. The sense or anti-sense strand of this hotspot vsiRNA, and the coldspot vsiRNA in both double-stranded and single-stranded formats possessed no activity in viral protection. We further demonstrated that the hotspot vsiRNA-mediated virus resistance had a threshold effect and required an active RDR6. These data show that hotspot vsiRNAs bear a functional significance on antiviral RNAi, suggesting that they may have the potential as an exogenous protection agent for controlling destructive viral diseases in plants.

In vitro and in vivo studies were conducted to investigate the potential of four biocontrol agents (BCAs), namely Bacillus megaterium, Pseudomonas fluorescens, Trichoderma viride, and T. harzianum, individually and in combination with sodium nitrophenolate (SN) to control marigold powdery mildew. The results showed that all treatments led to a significant inhibition in the conidial germination of Golovinomyces cichoracearum in vitro. Maximum inhibition was recorded by T. harzianum (1×10⁹ CFU mL^-1) + SN (1.5%), followed by T. viride + SN, and B. megaterium + SN at the same concentrations as follows: 83.6, 79.1, and 70.6%, respectively. While the lowest inhibition (20.4%) was recorded by P. fluorescens (1×10⁵ CFU mL^-1). In the greenhouse, all treatments applied significantly reduced the disease severity and the area under the disease progress curve (AUDPC). The combination treatments had a better disease control response than individual treatments. Similar results were achieved in the field, where disease severity reduced to 9.2 and 10.3% in plants treated with T. harzianum + SN in the first and second seasons, respectively, compared to 40.2 and 44.1% in control in both seasons. Likewise, AUDPC reduced to 274 and 315 in plants treated with T. harzianum + SN in the first and second seasons, respectively, compared to 1207 and 1340 in control in both seasons. The treatments improved growth and productivity characteristics, as well as photosynthetic pigments, total phenolic compounds (TPC), and polyphenol oxidase (PPO) activity, while significantly reducing free proline (FP). In conclusion, BCAs applied individually or in combination with SN can be used effectively to suppress powdery mildew of marigold.