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Daunorubicin (DNR) is an anthracycline antibiotic originating from soil-dwelling actinobacteria extensively used to treat malignant tumors. Over the decades, extensive attempts were made to enhance the production of anthracyclines by introducing genetic modifications and mutations in combination with media optimization, but the target production levels remain comparatively low. Developing an appropriate culture medium to maximize the yield of DNR and preventing autotoxicity for the producing organism remains a challenge. Our prospective review sheds light on a method involving perturbation that enhances the precursors to regulate the type II PKS pathway, enhancing cells’ capacity to increase secondary metabolite production. The suggested method also entails the preparation of culture media for the cultivation of Streptomyces sp. and enhanced yield of DNR, as well as making it inactive with iron or its reduced forms following efflux from the producer. The iron or iron–DNR complex is encapsulated by oleic acid or lipid micelle layers in the culture media, finally resulting in the generated inactive DNR and the DNR–iron–oil complex. This idea has the potential to protect the producer organism from autotoxicity and prevent the inhibition of metabolite production. The approach of substituting sugar with oil in culture media has a dual role wherein it promotes Streptomyces growth by utilizing lipids as an energy source and encapsulating the generated DNR–iron complex in the medium. In this review, we discussed aspects like anthracycline producers, biosynthesis pathways, and gene regulation; side effects of DNR; mechanisms for autotoxicity evasion; and culture media components for the enhancement of DNR production in Streptomyces sp. We anticipate that our work will help researchers working with secondary metabolites production and decipher a methodology that would enhance DNR yield and facilitate the extraction of the resulting DNR by lowering costs in large-scale fermentation.

Low-temperature plasma (LTP) is currently one of the non-invasive and environmentally friendly methods of seed treatment and is massively tested on various types of crops. For the needs of gene banks, the use of LTP technology represents the treatment of seeds before sowing to improve the germination and emergence of long-term stored seed samples. Seeds of four genotypes of wheat, oats, flax, and rapeseed stored in the gene bank for 1, 10, and 20 years were plasma treated for 20, 25, and 30 min. Standard germination parameters (SG3, SG7, GR, MGT, and GI), as well as predictive models, were used to evaluate the effect of plasma treatment on seeds, and the effect on seed metabolism was assessed by superoxide dismutase (SOD) activity. The plasma treatment had different effects on germination and on the enzymatic activity of the tested species, and the result was influenced by both the duration of the treatment and the crop species/genotype. The plasma treatment has a positive effect on germination parameters in flax and rapeseed; in some variants, as in wheat, oats generally reacted negatively. SOD activity was variable in wheat, while higher activity with increasing treatment time was found in other crops. The results of this first study focused on long-term stored seeds and showed the potential of plasma treatment of seeds of plant genetic resources, the possibility of stimulating the germination of stored PGRs, and the need to optimize treatment conditions for individual genotypes.

The gliding arc cold plasma treatment of wheat seeds is an alternative to chemical seed dressing, but this treatment is not very effective. This study was focused on testing the addition of salt, water and nitrogenous compounds during plasma treatment to increase the efficiency of the plasma seed treatment. The additives were not supplied in the gaseous state, as usual, but in an aqueous solution by spraying under a plasma nozzle during the treatment. The phytosanitary effect of the plasma treatment was evaluated based on the germination of Tilletia caries spores from artificially infected seeds on water agar. The viability of the seeds, growth and yield characteristics of the plasma-treated seeds were evaluated in the laboratory, greenhouse and even in field experiments with the primary goal of reducing spore vitality. The germination of T. caries spores was mostly limited to the variant with the addition of water during plasma treatment, where spore germination reached only 32.7% of the control variant while maintaining sufficient growth properties. The experiment demonstrated the suitability of using the addition of various substances during plasma treatment in the form of aqueous solutions and increasing the effectiveness of this treatment.

Members of the genus Armillaria are widespread forest pathogens against which effective protection has not yet been developed. Due to their longevity and the creation of large-scale cloning of Armillaria individuals, the use of mycoviruses as biocontrol agents (BCAs) against these pathogens could be an effective alternative. This work describes the detection and characterization of viruses in Armillaria spp. collected in the Czech Republic through the application of stranded total RNA sequencing. A total of five single-stranded RNA viruses were detected in Armillaria ostoyae and A. cepistipes, including viruses of the family Tymoviridae and four viruses belonging to the recently described “ambivirus” group with a circular ambisense genome arrangement. Both hammerhead (HHRz) and hairpin (HpRz) ribozymes were detected in all the ambiviricot sequences. Armillaria viruses were compared through phylogenetic analysis and confirmed their specific host by direct RT-PCR. One virus appears to infect both Armillaria species, suggesting the occurrence of interspecies transmission in nature.

In the past few years, huge economic and environmental losses have resulted by fungal pathogens in agriculture and food production worldwide. Fungal toxins also pose a threat for food production and animal health. In the last decade, the site-specific fungicides has been used in plant protection, providing highly efficient protection against phytopathogens. However, the use of pesticides boosted problems with resistance in the targeted organisms and increased negative environmental impact of insecticides, herbicides and fungicides. Therefore, new methods are needed to control resistant populations of plant pathogens, including fungi. Mycoviruses are fungal viruses, i.e. viruses infecting fungi. Some mycoviruses affect the growth and virulence of the host fungus and may prevent successful infection of plant host. They have potential to reduce phytopathogens. In this review, the successful examples how mycoviruses alter hypovirulence in well-known phytopathogens are discussed. Mycoviruses could be useful as a one of new next generation pest-control products.

We introduced here the first loop mediated isothermal amplification (LAMP) assay for the identification of honey fungus, Armillaria ostoyae, a basidiomycote playing an important role in spruce declines in the Palaearctic region. In total, 101 isolates, representing three Armillaria species, were used to develop a new LAMP assay to determine species specific identification. We have here described LAMP primers enhanced with fluorescent dye that are able to amplify A. ostoyae DNA and detect fungi in a fast single step reaction. The detection limit of LAMP was 1 pg of genomic DNA per reaction. We optimized a new LAMP assay for the rapid detection of A. ostoyae using the translation elongation factor 1-α (tef1) marker and fluorescence labelled oligonucleotide assimilating probe. The LAMP assay does not require any specialized equipment, hence it can be used in the field for the rapid detection of A. ostoyae even using the portable and mobile device. The specificity of the assay was confirmed by the use of A. ostoyae strains and Armillaria cepistipes and Armillaria gallica strains, respectively. In conclusion, the assay could be a rapid, specific, sensitive and low-cost tool for identification of A. ostoyae as well as the first step for expansion of this method in practical applications.

Microsatellite markers were used for the assessment of genetic diversity and genetic structure in a germplasm collection of yellow mustard, Sinapis alba L. The comprehensive collection of genetic resources represented 187 registered varieties, landraces, and breeding materials. Microsatellites generated 44 polymorphic alleles in 15 loci. Eleven of them were medium to highly polymorphic, and the high levels of observed heterozygosity (0.12–0.83) and Nei’s gene diversity index (0.11–0.68) indicated a high level of polymorphism. Based on PCoA and neighbor joining analyses, the genetic resources were divided into two groups. The range of genetic dissimilarity in the analysed collection was in the range of 0.00–1.00. The high level of dissimilarity between the accessions was documented by the high WAM value (33.82%). Bayesian clustering algorithms were performed in the STRUCTURE 2.3.4 software. The number of clusters was estimated at K = 2. The accessions were classified according to Q1/Q2 values. The low average values of the parameters Fst_1 (0.3482), Fst_2 (0.1916), and parameter alpha (0.0602) indicated substantial mating barriers between varieties and reproductive isolation due to the limited exchange of genetic resources between breeders. These results demonstrated the importance of extensive collections of genetic resources for the maintenance of genetic diversity and indicated considerable genetic differentiation among accessions.

The opium poppy’s ability to produce various alkaloids is both useful and problematic. Breeding of new varieties with varying alkaloid content is therefore an important task. In this paper, the breeding technology of new low morphine poppy genotypes, based on a combination of a TILLING approach and single-molecule real-time NGS sequencing, is presented. Verification of the mutants in the TILLING population was obtained using RT-PCR and HPLC methods. Only three of the single-copy genes of the morphine pathway among the eleven genes were used for the identification of mutant genotypes. Point mutations were obtained only in one gene (CNMT) while an insertion was obtained in the other (SalAT). Only a few expected transition SNPs from G:C to A:T were obtained. In the low morphine mutant genotype, the production of morphine was decreased to 0.1% from 1.4% in the original variety. A comprehensive description of the breeding process, a basic characterization of the main alkaloid content, and a gene expression profile for the main alkaloid-producing genes is provided. Difficulties with the TILLING approach are also described and discussed.

Cotyledons of 201 microspore-derived oilseed rape (Brassica napus) embryos were analysed for the presence of the cytoplasmic male sterility Shaan 2A restorer gene using the primer pair 3F/4R. In total, 172 plants regenerated and were grown to the generative stage. Among phenotypically non-haploid flowering plants, there was a broad spectrum of different flower types from normally developed flowers to flowers with short anthers, stamens, or deformed flowers with atypically developed floral organs, often with traces of pollen. For this reason, only 115 typically fertile or sterile accessions were selected for further phenotype-genotype comparisons. In 14 plants (12.2%) the phenotype did not match the genotype: two plants were marked as fertile, but without the Rf gene. Vice versa, in 12 sterile plants the Rf gene was determined. Nevertheless, the two fertile plants did not produce any pods after self-pollination. In total, 47 doubled haploid (DH) lines with good seed yield, carrying the Rf gene were harvested.

Western honey bee (Apis mellifera) is one of the most important pollinators in the world. Thus, a recent honey bee health decline and frequent honey bee mass losses have drawn attention and concern. Honey bee fitness is primarily reduced by pathogens, parasites, and viral load, exposure to pesticides and their residues, and inadequate nutrition from both the quality and amount of food resources. This study evaluated the prevalence of the most common honey bee pathogens and viruses in different habitats across the Czech Republic. The agroecosystems, urban ecosystems, and national park were chosen for sampling from 250 colonies in 50 apiaries. Surprisingly, the most prevalent honey bee pathogens belong to the family Trypanosomatidae including Lotmaria passim and Crithidia mellificae. As expected, the most prevalent viruses were DWV, followed by ABPV. Additionally, the occurrence of DWV-B and DWV-C were correlated with honey bee colony mortality. From the habitat point of view, most pathogens occurred in the town habitat, less in the agroecosystem and least in the national park. The opposite trend was observed in the occurrence of viruses. However, the prevalence of viruses was not affected by habitat.

Water deficiency is one of the most significant abiotic stresses that negatively affects growth and reduces crop yields worldwide. Most research is focused on model plants and/or crops which are most agriculturally important. In this research, drought stress was applied to two drought stress contrasting varieties of Papaver somniferum (the opium poppy), a non-model plant species, during the first week of its germination, which differ in responses to drought stress. After sowing, the poppy seedlings were immediately subjected to drought stress for 7 days. We conducted a large-scale transcriptomic and proteomic analysis for drought stress response. At first, we found that the transcriptomic and proteomic profiles significantly differ. However, the most significant findings are the identification of key genes and proteins with significantly different expressions relating to drought stress, e.g., the heat-shock protein family, dehydration responsive element-binding transcription factors, ubiquitin E3 ligase, and others. In addition, metabolic pathway analysis showed that these genes and proteins were part of several biosynthetic pathways most significantly related to photosynthetic processes, and oxidative stress responses. A future study will focus on a detailed analysis of key genes and the development of selection markers for the determination of drought-resistant varieties and the breeding of new resistant lineages.

Abstract Background Non-native species have aroused scientific interest because of their ability to successfully colonise areas to which they have been introduced, despite their sometimes limited genetic variation compared to their native range. These species establish themselves with the aid of some pre-existing features favouring them in the new environment. Pectinatella magnifica (Leidy, 1851), the freshwater magnificent bryozoan, is non-native in Europe and Asia. This study was designed to determine the genetic diversity and population structure of P. magnifica colonies collected from the Protected Landscape Area (PLA) and UNESCO Biosphere Reserve Třeboňsko (the Czech Republic) in the 2009 and 2011–2014 periods using Amplified Fragment Length Polymorphism (AFLP). Findings The vast majority of the examined non-native colonies, except three colonies sampled in 2012, expressed very low levels of genetic variation, not differentiating from the USA native colony. The Bayesian clustering approach grouped the 28 accessions into two genetically different populations. Conclusions The data suggest relatively low gene diversity within all colonies, which might reflect the recent expansion of P. magnifica in the Czech Republic.

Malnutrition is one of the main problems related to the global mass collapse of honey bee colonies, because in honey bees, malnutrition is associated with deterioration of the immune system and increased pesticide susceptibility. Another important cause of mass bee colonies losses is the use of pesticides. Therefore, the goal of this study was to verify the influence of polyphenols on longevity, food consumption, and cytochrome P450 gene expression in worker bees intoxicated by thiacloprid. The tests were carried out in vitro under artificial conditions (caged bees). A conclusively lower mortality rate and, in parallel, a higher average food intake, were observed in intoxicated bees treated using a mixture of phenolic acids and flavonoids compared to untreated intoxicated bees. This was probably caused by increased detoxification capacity caused by increased expression level of genes encoding the cytochrome P450 enzyme in the bees. Therefore, the addition of polyphenols into bee nutrition is probably able to positively affect the detoxification capacity of bees, which is often reduced by the impact of malnutrition resulting from degradation of the environment and common beekeeping management.

Ascosphaera apis is a causative agent of chalkbrood, which is one of the most widespread honey bee diseases. In our experiments, the influence of several artificial media and cultivation under different temperatures was evaluated. Concretely, the radial growth of separated mating types was measured, reproductive structures in a Neubauer hemocytometer chamber were counted simultaneously, and the morphometry of spore cysts and spore balls was assessed. The complex set of experiments determined suitable cultivation conditions. A specific pattern between reproductive structure size and temperature was found. The optimal temperature for both mating types was 30 °C. SDA and YGPSA media are suitable for fast mycelial growth. Moreover, the effect of bee brood on fungus growth and development in vitro was investigated by modification of culture medium. The newly modified medium PDA-BB4 was most effective for the production of the reproductive structures. The result suggests that honey bee brood provides necessary nutrients for proper fungus development during in vitro cultivation. As there is no registered therapeutic agent against chalkbrood in most countries, including the European Union, the assessment of A. apis growth and development in different conditions could help to understand fungus pathogenesis and thus control chalkbrood disease.

Xanthomonas gardneri is one of the causal agents of bacterial spot (BS), an economically important bacterial disease of tomato and pepper. Field-deployable and portable loop-mediated isothermal amplification (LAMP)-based instruments provide rapid and sensitive detection of plant pathogens. In order to rapidly and accurately identify and differentiate X. gardneri from other BS-causing Xanthomonas spp., we optimized a new real-time monitoring LAMP-based method targeting the X. gardneri-specific hrpB gene. Specificity and sensitivity of real-time and colorimetric LAMP assays were tested on the complex of bacterial strains pathogenic to tomato and pepper and on plants infected by the pathogen. The assay detection limit was 1 pg/μL of genomic DNA with an assay duration of only 30 min. The use of portable and handheld instruments allows for fast analysis, reducing the diagnosis time, and can contribute to proper disease management and control of X. gardneri. Due to the high efficiency of this method, we suggest its use as a standard diagnostic tool during phytosanitary controls.

Plasma treatment is an interesting technology with many potential applications in industry. At present, more and more often we encounter papers that deal with utilization of plasma technology in food industry. This mini-review provides an overview of the latest developments in this area. At the beginning the principles of plasma interaction with solid surfaces are shortly discussed. The next chapters are focused on sterilization of food from bacteria and fungi. The infl uence of plasma treatment on the food quality and nutritional parameters is discussed in last section.

Background: Microspore embryogenesis and cytoplasmic male sterility system (CMS) are two approaches widely exploited in Brassica napus breeding for production of homozygous doubled haploid (DH) lines and F1 hybrids respectively. Cytoplasmic male sterility system (CMS) is one of the most important pollination systems for hybrid seed production and utilisation of doubled haploid system to quickly prepare fully homozygous fertility restorer lines for CMS Ogu-INRA is very beneficial. Generally, only a small part of microspore-derived embryos is used for plant regeneration, without any knowledge about their properties. Therefore, the possibility of early detection of desirable genotypes bearing a single dominant nuclear fertility restorer (Rfo) gene, can double the success of selection and reduce the production costs. Results: To maximize the efficiency and yield of regenerated microspore-derived embryos (MDEs) with the Rfo gene, a protocol for reliable and early, non-destructive selection of desired MDE genotypes was developed. The total amount of 636 cotyledonary embryos was tested by PCR, out of which 37% (237/636) were shown to bear the Rfo gene (instead of 50% according to the expected 1:1 segregation ratio for a single copy gene) and 218 of these fertility restorer plants were fully grown to flowering stage. New molecular marker has been demonstrated to have 100% of co-segregation with the phenotypic evaluation. Conclusion: Technique developed in this study provides early and non-destructive sampling of embryonic tissue and the use of new markers for simple and efficient control of the presence of Rfo gene in all accessions.

ABSTRACT: In recent decades, interest in non-traditionally colored eggs has increased. For breeders, this market interest means breeding lines of laying hens that lay eggs of varied colors, such as the blue-green eggshells (Dominant Greenshell) in this study. This study presents the results of genotyping the polymorphism of the O locus responsible for shell coloration and photometric measurement of eggshell color based on the CIELAb system, which was carried out on the unique Czech breeding population Dominant Greenshell. The aim was to use a combination of phenotyping using the CIELab System method and genotyping of the O locus using the end-point PCR approach with the main focus on the accuracy of distinguishing shell color genotypes, streamlining the selection of dominant homozygotes in the O locus, optimizing this technology for the most efficient and cost-effective selection procedure in practical hen breeding. The optometric method was able to reliably distinguish only dominant and recessive phenotypes and eliminate from the population only undesirable recessive homozygotes with a white colored shell. The parameter a* (redness/greenness) from the CIELab color space turned out to be absolutely key for distinguishing dominant and recessive phenotypes. Using the CART methodology, a classification tree built on discriminating optometric characteristics a-blunt was obtained, however, for the group of desirable O/O homozygotes, the selection approach would result in incorrect genotyping of 31% of individuals. Therefore, a combined approach based on rapid and simple elimination of recessive homozygotes using phenotyping (CIELab photometric measurement) and molecular identification of the EAV-HP insertion in the SLCO1B3 gene in dominant phenotypes, regardless of color intensity affected by laying time/order, and allowing reliable elimination, has proven to be the most effective method to distinguish heterozygotes from the breeding population. The combination of optometric and molecular selection methods then leads to more efficient selection, reduction of overall selection costs. This process led to the stabilization of the breeding population within one generation and the achievement of a pure homozygous line with regard to eggshell color.

Marker-assisted selection based on fast and accurate molecular analysis of individual genes is considered an acceptable tool in the speed-up of the genetic improvement of production performance in chickens. The objective of this study was to detect the single nucleotide polymorphisms (SNPs) in the IGF1, IGFBP2 and TGFß3 genes, and to investigate their associations with growth performance (body weight (BW) and average daily gain (ADG) at 14, 21, 28, 35 and 42 days of age) and carcass traits in broilers. Performance (carcass) data (weight before slaughter; weights of the trunk, giblets, abdominal fat, breast muscle and thigh muscle; slaughter value and slaughter percentage), as well as blood samples for DNA extraction and SNP analysis, were obtained from 97 chickens belonging to two different lines (Hubbard F15 and Cobb E) equally divided between the two sexes. The genotypes were detected using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) methods with specific primers and restrictase for each gene. The statistical analysis discovered significant associations (p < 0.05) between the TGFβ3 SNP and the following parameters: BW at 21, 28 and 35 days, trunk weight and slaughter value. Association analysis of BWs (at 21, 28 and 35 days) and SNPs was always significant for codominant, dominant and overdominant genetic models, showing a possible path for genomic selection in these chicken lines. Slaughter value was significant for codominant, recessive and overdominant patterns, whereas other carcass traits were not influenced by SNPs. Based on the results of this study, we suggested that the TGFβ3 gene could be used as a candidate gene marker for chicken growth traits in the Hubbard F15 and Cobb E population selection programs, whereas for carcass traits further investigation is needed.