Habituation to a Deterrent Plant Alkaloid Develops Faster in the Specialist Herbivore Helicoverpa assulta Than in Its Generalist Congener Helicoverpa armigera and Coincides with Taste Neuron Desensitisation

The two closely related moth species, Helicoverpa armigera and H. assulta differ strongly in their degree of host-plant specialism. In dual-choice leaf disk assays, caterpillars of the two species that had been reared on standard artificial diet were strongly deterred by the plant-derived alkaloid strychnine. However, caterpillars of both species reared on artificial diet containing strychnine from neonate to the 5th instar were insensitive to this compound. Fifth instar caterpillars of H. assulta and 4th or 5th instars of H. armigera not exposed to strychnine before were subjected to strychnine-containing diet for 24 h, 36 h, 48 h, or 72 h. Whereas H. assulta displayed habituation to strychnine after 48 h, it took until 72 h for H. armigera to become habituated. Electrophysiological tests revealed that a deterrent-sensitive neuron in the medial sensillum styloconicum of both species displayed significantly reduced sensitivity to strychnine that correlated with the onset of habituation. We conclude that the specialist H. assulta habituated faster to strychnine than the generalist H. armigera and hypothesis that desensitization of deterrent-sensitive neurons contributed to habituation.

Direct induction of adventitious root in Schefflera octophylla (Lour.) Harms from leaf explants cultured in vitro

Schefflera octophylla (Lour.) Harms is a precious plant species belonging to the Araliaceae family. All parts of plant have been used to create products for human health. In tissue culture of medicinal plants, the induction and multiplication of adventitious root of Schefflera octophylla for biomass collection have been studied. In this report, results on induction of adventitious root from leaf explants cultured in vitro of this plant species were presented. Leaf disks (~ 10 x 10 mm), leaf transverse - thin cell layers (t-TCLs) (~ 3 x 10 mm) were cultured on different mineral media MS, ½MS, B5, SH with NAA (0 - 5 mg/L), sucrose (0 - 50 g/L) and light intensity (0 - 4,000 lux). The results showed that, 30 days after culturing on ½MS solid medium plus 3 mg/L NAA, and 30 g/L sucrose in 4,000 lux light condition, direct formation of adventitious root was best from leaf disks, t-TCLs with rooting rate (%) 100, 100; root number/sample 68.80, 21.96; root lenght (mm) 16.53, 15.53, respectively. Leaf disk culture resulted in better rooting than t-TCL culture in two criteria of root number and root length. Morphological and histological observations of adventitious root primordia formation in the leaf disk were also performed. This is the first report on direct formation of adventitious root by in vitro culture of leaf disks/t-TCLs in Schefflera octophylla with very high efficiency, creating basis for further studies on root biomass multiplication for production of bioactive compounds.

High-Throughput Feeding Bioassay for Lepidoptera Larvae

Finding plant cultivars that are resistant or tolerant against lepidopteran pests, takes time, effort and is costly. We present here a high throughput leaf-disk consumption assay system, to screen plants for resistance or chemicals for their deterrence. A webcam capturing images at regular intervals can follow the feeding activities of 150 larvae placed into individual cages. We developed a computer program running under an open source image analysis program to analyze and measure the surface of each leaf disk over time. We further developed new statistical procedures to analyze the time course of the feeding activities of the larvae and to compare them between treatments. As a test case, we compared how European corn borer larvae respond to a commercial antifeedant containing azadirachtin, and to quinine, which is a bitter alkaloid for many organisms. As expected, increasing doses of azadirachtin reduced and delayed feeding. However, quinine was poorly effective at the range of concentrations tested (10–5 M to 10–2 M). The model cage, the camera holder, the plugins, and the R scripts are freely available, and can be modified according to the users’ needs.

A semi-automated design for high-throughput Lepidoptera larvae feeding bioassays

Lepidopteran pests cause considerable damage to all crops over the world. As larvae are directly responsible for these damages, many research efforts are devoted to find plant cultivars which are resistant against them. However, such studies take time, efforts and are costly, especially when one wants to not only find resistance traits but also evaluate their heritability. We present here a high throughput approach to screen plants for resistance or chemicals for their deterrence, using a leaf-disk consumption assay, which is both suitable for large scale tests and economically affordable. To monitor larvae feeding on leaf disks placed over a layer of agar, we designed 3D models of 50 cages arrays. One webcam can sample simultaneously 3 of such arrays at a rate of 1 image/min, and follow individual feeding activities in each cage as the movements of 150 larvae. The resulting image stacks are first processed with a custom program running under an open-source image analysis package (Icy) to measure the surface of each leaf disk over time. We further developed statistical procedures running under the R package, to analyze the time course of the feeding activities of the larvae and to compare them between treatments. As a test case, we compared how European corn borer larvae respond to quinine, considered as a bitter alkaloid for many organisms, and to Neemazal containing azadirachtin, which is a common antifeedant against pest insects. We found that increasing doses of azadirachtin reduce and delay feeding. However, contrary to our expectation, quinine was found poorly effective at the range of concentrations tested. The 3D printed model of the cage, of the camera holder, the plugins running under Icy, and the R procedures are freely available, and can be modified according to the particular needs of the users.

Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with other techniques

Background Tomato brown rugose fruit virus (ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (Solanum lycopersicum L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin. Methods Molecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan minor groove binder probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extraction, direct crude extract and leaf-disk crude extract) were evaluated. Results The Sicilian isolate ToB-SIC01/19 (6,391 nt) showed a strong sequence identity with the isolates TBRFV-P12-3H and TBRFV-P12-3G from Germany, Tom1-Jo from Jordan and TBRFV-IL from Israel. The ToB-SIC01/19 isolate was successfully transmitted by mechanical inoculations in S. lycopersicum L. and Capsicum annuum L., but no transmission occurred in S. melongena L. The developed real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the open reading frames3, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus Tobamovirus, minimising false-negative results. Using immunocapture and total RNA extraction procedures, the real-time RT-PCR and end-point RT-PCR gave the same comparable results. Using direct crude extracts and leaf-disk crude extracts, the end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive real-time RT-PCR assay will be a particularly valuable tool for early ToBRFV diagnosis, optimising procedures in terms of costs and time.

A High-Throughput Phenotyping System Using Machine Vision to Quantify Severity of Grapevine Powdery Mildew

Powdery mildews present specific challenges to phenotyping systems that are based on imaging. Having previously developed low-throughput, quantitative microscopy approaches for phenotyping resistance to Erysiphe necator on thousands of grape leaf disk samples for genetic analysis, here we developed automated imaging and analysis methods for E. necator severity on leaf disks. By pairing a 46-megapixel CMOS sensor camera, a long-working distance lens providing 3.5× magnification, X-Y sample positioning, and Z-axis focusing movement, the system captured 78% of the area of a 1-cm diameter leaf disk in 3 to 10 focus-stacked images within 13.5 to 26 seconds. Each image pixel represented 1.44 μm2 of the leaf disk. A convolutional neural network (CNN) based on GoogLeNet determined the presence or absence of E. necator hyphae in approximately 800 subimages per leaf disk as an assessment of severity, with a training validation accuracy of 94.3%. For an independent image set the CNN was in agreement with human experts for 89.3% to 91.7% of subimages. This live-imaging approach was nondestructive, and a repeated measures time course of infection showed differentiation among susceptible, moderate, and resistant samples. Processing over one thousand samples per day with good accuracy, the system can assess host resistance, chemical or biological efficacy, or other phenotypic responses of grapevine to E. necator. In addition, new CNNs could be readily developed for phenotyping within diverse pathosystems or for diverse traits amenable to leaf disk assays.

Investigating Phenylamide Insensitivity in Wisconsin Populations of Pseudoperonospora humuli

Hop downy mildew caused by the oomycete Pseudoperonospora humuli is primarily managed with fungicides because commercial varietal resistance is unavailable. Mefenoxam, a phenylamide-class fungicide, is highly effective for systemic disease control but is at high risk for the development of pathogen insensitivity. Due to the recent expansion of hop production in Wisconsin, it was unknown if P. humuli could be managed with mefenoxam because insensitivity has been documented in other regions. During 2015 to 2017, isolates of P. humuli were collected from commercial yards throughout Wisconsin. Isolates were screened for insensitivity using a leaf disk assay with fungicide-amended water agar. Insensitivity was determined using a single discriminatory dose of 25 μg/ml of mefenoxam, and isolates were considered insensitive if they exhibited at least 50% sporulation on amended media relative to nonamended water agar. Over all years, nearly 48% of all isolates were insensitive to mefenoxam. The incidence of insensitivity varied between individual hop yards, with most yards exhibiting a mixture of sensitive and insensitive isolates. At this time, mefenoxam-insensitive populations do not appear to be predominant in Wisconsin. Growers should monitor the effectiveness of mefenoxam applications in their hop yards and be responsive to changes in crop response.