Reference genes are commonly used as the endogenous normalisation measure for the relative quantification of target genes. The appropriate application of quantitative real-time PCR (RT-qPCR), however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. For this purpose, seven reference genes were investigated in tissues of the most important cereals (wheat, barley and oats). Titre of Barley yellow dwarf virus (BYDV) was determined in oats using relative quantification with different reference genes and absolute quantification, and the results were compared.

Heat stress is a frequent environmental constraint. Phytohormones can significantly affect plant thermotolerance. This study compares the effects of exogenous cytokinin meta-topolin-9-(tetrahydropyran-2-yl)purine (mT9THP) on rice (Oryza sativa) under control conditions, after acclimation by moderate temperature (A; 37 °C, 2h), heat stress (HS; 45 °C, 6h) and their combination (AHS). mT9THP is a stable cytokinin derivative that releases active meta-topolin gradually, preventing the rapid deactivation reported after exogenous cytokinin application. Under control conditions, mT9THP negatively affected jasmonic acid in leaves and abscisic and salicylic acids in crowns (meristematic tissue crucial for tillering). Exogenous cytokinin stimulated the emission of volatile organic compounds (VOC), especially 2,3-butanediol. Acclimation upregulated trans-zeatin, expression of stress- and hormone-related genes, and VOC emission. The combination of acclimation and mT9THP promoted the expression of stress markers and antioxidant enzymes and moderately increased VOC emission, including 2-ethylhexyl salicylate or furanones. AHS and HS responses shared some common features, namely, increase of ethylene precursor aminocyclopropane-1-carboxylic acid (ACC), cis-zeatin and cytokinin methylthio derivatives, as well as the expression of heat shock proteins, alternative oxidases, and superoxide dismutases. AHS specifically induced jasmonic acid and auxin indole-3-acetic acid levels, diacylglycerolipids with fewer double bonds, and VOC emissions [e.g., acetamide, lipoxygenase (LOX)-derived volatiles]. Under direct HS, exogenous cytokinin mimicked some positive acclimation effects. The combination of mT9THP and AHS had the strongest thermo-protective effect, including a strong stimulation of VOC emissions (including LOX-derived ones). These results demonstrate for the first time the crucial contribution of volatiles to the beneficial effects of cytokinin and AHS on rice thermotolerance.

Barley yellow dwarf virus (BYDV) causes an often-devastating disease of cereals that is most effectively controlled by using plant genotypes that are resistant or tolerant to the virus. New barley lines Vir8:3 and Vir13:8, with pyramided resistance genes against different pathogens and resistance gene Ryd2 against BYDV, are currently being tested. Because microRNAs (miRNAs) are associated with antiviral plant defense, here we compared the miRNA profiles in these lines and in cultivar Wysor (carrying one resistance gene, Ryd2), with and without BYDV infection and after feeding by virus-free aphids, to determine whether the miRNA profile in the resistant variety bear similarities with the newly developed lines. The BYDV titer for each group was also determined and compared to the titer in sensitive cultivar Graciosa. Among 746 miRNAs identified in barley, 66 were known miRNAs, and 680 were novel. The expression of 73 miRNAs differed significantly after BYDV infection, including the strong, specific upregulation of novel miRNA10778 that was conserved across all the barley genotypes. This miRNA belongs to the H box and ACA box (H/ACA) snoR14 family of RNAs (Rf01280) and is associated with pseudourydilation. The expression of 48 miRNAs also differed depending on the barley genotype. The profile of miRNAs expressed in Vir8:3 and Vir13:8 in response to BYDV was similar and differed from that of Wysor. Insights into the expression patterns of miRNAs in response to BYDV in barley provided here will benefit further studies toward understanding the resistance mechanisms and developing novel strategies against virus infections.

The bird cherry-oat aphid (Rhopalosiphum padi (L.)) is one of the most detrimental pests of cereals, causing harm mainly by transmitting Barley yellow dwarf virus (BYDV). R. padi migration has been monitored since 1992 using suction traps at five sites in the Czech Republic. The count data were subjected to the following different analyses: (i) the minimum temperature thresholds for the aphids to take off were determined; (ii) a partial redundancy analysis using the minimum, average, and maximum temperatures, as well as the wind speed, the precipitation total, and past aphid migration descriptors was performed to explain the relationship between aphid occurrences and weather patterns; and (iii) three types of models from the field of machine learning were used to predict aphid occurrences. According to our findings, (i) in Central Europe, 8°C is the temperature threshold for R. padi migration unless insufficient daylight delays the take-off; (ii) weather conditions occurring roughly 9 months before R. padi migration influence the migration size; (iii) the duration of the summer migration influences the autumn migration size; and (iv) the daytime and nighttime temperatures in the autumn determine the summer peak, whereas winter frosts and precipitation influence the autumn peak.

A challenge in bee protection is to assess the risks of pesticide-pathogen interactions. Lotmaria passim, a ubiquitous unicellular parasite in honey bees, is considered harmful under specific conditions. Imidacloprid causes unpredictable side effects. Research indicates that both L. passim and imidacloprid may affect the physiology, behavior, immunity, microbiome and lifespan of honey bees. We designed cage experiments to test whether the infection of L. passim is affected by a sublethal dose of imidacloprid. Workers collected at the time of emergence were exposed to L. passim and 2.5 μg/L imidacloprid in the coexposure treatment group. First, samples of bees were taken from cages since they were 5 days old and 3 days postinfection, i.e., after finishing an artificial 24 h L. passim infection. Additional bees were collected every two additional days. In addition, bees frozen at the time of emergence and collected from the unexposed group were analyzed. Abdomens were analyzed using qPCR to determine parasite load, while corresponding selected heads were subjected to a label-free proteomic analysis. Our results show that bees are free of L. passim at the time of emergence. Furthermore, imidacloprid considerably increased the prevalence as well as parasite loads in individual bees. This means that imidacloprid facilitates infection, enabling faster parasite spread in a colony and potentially to surrounding colonies. The proteomic analysis of bee heads showed that imidacloprid neutralized the increased transferrin 1 expression by L. passim. Importantly, this promising marker has been previously observed to be upregulated by infections, including gut parasites. This study contributes to understanding the side effects of imidacloprid and demonstrates that a single xenobiotic/pesticide compound can interact with the gut parasite. Our methodology can be used to assess the effects of different compounds on L. passim.

Aims Endoluminal radiofrequency ablation (RFA) has been suggested a promising treatment modality for palliative treatment of cholangiocarcinoma (CCC) and pancreatic cancer (PC). Methods In a randomized controlled manner, we compared endoluminal radiofrequency ablation plus stenting with stenting alone in patients with malignant biliary obstruction with survival as the primary outcome and stent patency as the secondary outcome. Results A total of 161 patients with malignant biliary obstruction (85 with CCC and 76 with PC) were randomized [81 RFA + stent (44 CCC, 37 PC), 80 stent (41 CCC, 39 PC)]. The recruitment was terminated for futility after an interim analysis before enrolling the originally planned 280 patients. There was no difference in survival in patients with cholangiocarcinoma (median survival 10.5 months (95%CI 6.7-18.3) vs. 10.6 months (95%CI 9.0-24.8) and pancreatic cancer (median survival 6.4 months (95%CI 4.3-9.7) vs. 5.6 months (95% CI 5.6-11.3). Furthermore, no benefit was seen in stent patency in both CCC patients and PC patients. Complications occurred in 7 patients (8.6%) treated with RFA and 7 patients (8.8%) treated with stenting only (NS) ([Table 1]). Conclusions In malignant biliary obstruction, endoluminal radiofrequency ablation in addition to stenting was not superior to stenting alone in prolonging survival or stent patency. Article published online: 14 April 2023 © 2023. European Society of Gastrointestinal Endoscopy. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany