The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics.

The brown planthopper (Nilaparvata lugens, BPH) is a destructive insect pest posing a serious threat to rice production. The fungal entomopathogen Metarhizium anisopliae is a promising alternative that can be used for BPH biocontrol. Recent studies have highlighted the significant involvement of gut microbiota in the insect–fungus interactions. In the presented study, we investigated the effects of topical fungal infection on the gut microbial community structure in BPH. Our results revealed that topical infection with M. anisopliae increased the bacterial load and altered the bacterial community structure in the gut of BPH. The relative abundances of the dominant gut bacteria at the order, family and genus level were significantly different between fungus-infected and uninfected groups. At the genus level, the uninfected BPH harbored high proportions of Pantoea and Enterobacter in the gut, whereas the fungus-infected BPH gut was absolutely dominated by Acinetobacter. Moreover, topical fungal infection significantly inhibited the expressions of immune-related genes encoding anti-microbial protein and dual oxidase that were involved in the maintenance of gut microbiota homeostasis, indicating that gut bacteria imbalance might be attributed in part to the suppression of gut immunity caused by fungal pathogen. Our results highlighted the importance of the gut microbial community during interactions between fungal pathogens and insect hosts.

Cnaphalocrocis medinalis is a destructive migratory rice pest. Although many studies have investigated its behavioral and physiological responses to environmental changes and migration-inducing factors, little is known about its molecular mechanisms. This study was conducted to select suitable RT-qPCR reference genes to facilitate future gene expression studies. Here, thirteen candidate housekeeping genes (EF1α, AK, EF1β, GAPDH, PGK, RPL13, RPL18, RPS3, 18S rRNA, TBP1, TBP2, ACT, and UCCR) were selected to evaluate their stabilities under different conditions using the ∆CT method; the geNorm, NormFinder, BestKeeper algorithms; and the online tool RefFinder. The results showed that the most stable reference genes were EF1β, PGK, and RPL18, related to developmental stages; RPS3 and RPL18 in larval tissues; EF1β and PGK in larvae feeding on different rice varieties; EF1α, EF1β, and PGK in larvae temperature treatments; PGK and RPL13, related to different adult ages; PGK, EF1α, and ACT, related to adult nutritional conditions; RPL18 and PGK, related to adult mating status; and, RPS3 and PGK, related to different adult take-off characteristics. Our results reveal reference genes that apply to various experimental conditions and will greatly improve the reliability of RT-qPCR analysis for the further study of gene function in this pest.