Wilt disease of roselle (Hibiscus sabdariffa L.) is common in Taiwan; however, the causative agent remains unknown. The stems of wilted roselle are browned, slightly constricted, and covered by white aerial hyphae, suggesting that potential pathogens may originate from soil. To identify the potential pathogens, we conducted a rhizosphere microbiota survey in phenotypically healthy and diseased plants through fungal internal transcribed spacer (ITS) and bacterial 16S rRNA amplicon sequencing for uncovering the microbial compositions in the roselle rhizosphere. The fungal family Nectriaceae exhibited significantly higher abundance in diseased rhizospheres than in healthy rhizospheres, and this bacterial community was more specific to geography (i.e., plot-dependent) than to rhizosphere disease status. However, a few bacterial groups such as Bacilli were associated with the healthy rhizosphere. Fusarium species were the most dominant species of Nectriaceae in the survey and became the main target for potential pathogen isolation. We successfully isolated 119 strains from diseased plants in roselle fields. Koch’s postulates were used to evaluate the pathogenicity of these strains; our results indicated that Fusarium solani K1 (FsK1) can cause wilting and a rotted pith in roselles, which was consistent with observations in the fields. This is the first demonstration that F. solani can cause roselle wilt in Taiwan. Furthermore, these newly isolated strains are the most dominant operational taxonomic units detected in ITS amplicon sequencing in diseased rhizospheres, which serves as further evidence that F. solani is the main pathogen causing the roselle wilt disease. Administration of Bacillus velezensis SOI-3374, a strain isolated from a healthy roselle rhizosphere, caused considerable anti-FsK1 activity, and it can serve as a potential biocontrol agent against roselle wilt disease.

24Biomolecules that respond to different external stimuli enable the remote control of genetically 25 modified cells. Chemogenetics and optogenetics, two tools that can control cellular activities 26 via synthetic chemicals or photons, respectively, have been widely used to elucidate underlying 27 physiological processes. These methods are, however, very invasive, have poor penetrability, 28 or low spatiotemporal precision, attributes that hinder their use in therapeutic applications. We 29 report herein a sonogenetic approach that can manipulate target cell activities by focused 30 ultrasound stimulation. This system requires an ultrasound-responsive protein derived from an 31 engineered auditory-sensing protein prestin. Heterogeneous expression of mouse prestin 32 containing two parallel amino acid substitutions, N7T and N308S, that frequently exist in 33 prestins from echolocating species endowed transfected mammalian cells with the ability to 34 sense ultrasound. An ultrasound pulse of low frequency and low pressure efficiently evoked 35 cellular calcium responses after transfecting with prestin(N7T, N308S). Moreover, pulsed 36 ultrasound can also non-invasively stimulate target neurons expressing prestin(N7T, N308S) 37 in deep regions of mice brains. Our study delineates how an engineered auditory-sensing 38 protein can cause mammalian cells to sense ultrasound stimulation. Moreover, owing to the 39 great penetration of low-frequency ultrasound (~400 mm in depth), our sonogenetic tools will 40 serve as new strategies for non-invasive therapy in deep tissues of large animals like primates. 41 42 43 44 45 46

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