Development and implementation of a novel CAPS assay reveals high prevalence of a boscalid resistance marker and its co-occurrence with an azole resistance marker in Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs), are frequently used against powdery mildew (PM) fungi, such as Erysiphe necator, the causal agent of grapevine PM. Fungicide resistance, however, hinders effective control. DNA-based monitoring facilitates the recognition of resistance. We aimed (i) to adapt an effective method to detect a widespread genetic marker of resistance to boscalid, a commonly used SDHI, and (ii) to study the co-occurrence of the marker with a marker of resistance to demethylase inhibitor (DMI) fungicides. Sequencing of the sdhB gene identified a non-synonymous substitution, denoted as sdhB-A794G, leading to an amino acid change (H242R) in the sdhB protein. In vitro fungicide resistance tests showed that E. necator isolates carrying sdhB-A794G were resistant to boscalid. We adopted a cleaved amplified polymorphic sequence-based method and screened more than 500 field samples collected from five Hungarian wine regions in two consecutive years. The sdhB-A794G marker was detected in all wine regions and in both years, altogether in 61.7% of samples, including 20.5% in which both sdhB-A794G and the wild-type were present. The frequency of sdhB-A794G was higher in SDHI-treated vineyards than in vineyards without any SDHI application. A significant difference in the presence of the marker was detected among wine regions; its prevalence ranged from none to 100%. We identified significant co-occurrence of sdhB-A794G with the CYP51-A495T (Y136F) mutation of the CYP51 gene, a known marker of resistance to DMIs. The monitoring of fungicide resistance is fundamental for the successful control of E. necator. Our rapid, cost-effective diagnostic method will support decision-making and fungicide resistance monitoring and management.

Stabilization of G-Quadruplex Structures of the SARS-CoV-2 Genome by TMPyP4, BRACO19, and PhenDC3.

The G-quadruplex is one of the non-canonical structures formed by nucleic acids, which can be formed by guanine-rich sequences. They became the focus of much research when they were found in several oncogene promoter regions and also in the telomeres. Later on, they were discovered in viruses as well. Various ligands have been developed in order to stabilize DNA G-quadruplexes, which were believed to have an anti-cancer or antiviral effect. We investigated three of these ligands, and whether they can also affect the stability of the G-quadruplex-forming sequences of the RNA genome of SARS-CoV-2. All three investigated oligonucleotides showed the G-quadruplex form. We characterized their stability and measured their thermodynamic parameters using the Förster resonance energy transfer method. The addition of the ligands caused an increase in the unfolding temperature, but this effect was smaller compared to that found earlier in the case of G-quadruplexes of the hepatitis B virus, which has a DNA genome.

Fusarium Species Associated with Fusarium Head Blight in Hungarian Wheat Fields.

The species composition of the genus Fusarium associated with Fusarium head blight (FHB) in wheat fields of Hungary in the year 2019 was assessed. Symptomatic wheat heads were collected at 20 geographical locations representing different ecosystems. A total of 256 Fusarium strains were isolated and identified by partial sequences of the translation elongation factor 1-alpha gene and, where required, the second-largest subunit of the DNA-directed RNA polymerase gene. Overall, Fusarium graminearum (58.2%) proved to be the dominant species, followed by F. annulatum (formerly F. proliferatum) (17.2%) and F. verticillioides (7.4%). The presence of all other species, including F. culmorum, in the population was less than 5%. F. graminearum was identified as the main species associated with FHB at 14 sampling sites. Fumonisin-producing F. annulatum, primarily known as the pathogen of maize in Hungary, was detected nearly as frequently as F. graminearum at three locations and dominated at two other sites. F. poae was not found during the survey. F. vorosii, a species that is believed to be of Asian origin and was already found in Hungary in 2002, was identified at two locations.

Comprehensive analyses of the occurrence of a fungicide resistance marker and the genetic structure in Erysiphe necator populations.

Genetically distinct groups of Erysiphe necator, the fungus causing grapevine powdery mildew infect grapevine in Europe, yet the processes sustaining stable genetic differences between those groups are less understood. Genotyping of over 2000 field samples from six wine regions in Hungary collected between 2017 and 2019 was conducted to reveal E. necator genotypes and their possible differentiation. The demethylase inhibitor (DMI) fungicide resistance marker A495T was detected in all wine regions, in 16% of the samples. Its occurrence differed significantly among wine regions and grape cultivars, and sampling years, but it did not differ between DMI-treated and untreated fields. Multilocus sequence analyses of field samples and 59 in vitro maintained isolates revealed significant genetic differences among populations from distinct wine regions. We identified 14 E. necator genotypes, of which eight were previously unknown. In contrast to the previous concept of A and B groups, European E. necator populations should be considered genetically more complex. Isolation by geographic distance, growing season, and host variety influence the genetic structuring of E. necator, which should be considered both during diagnoses and when effective treatments are planned.

Multilocus molecular phylogenetic-led discovery and formal recognition of four novel root-colonizing Fusarium species from northern Kazakhstan and the phylogenetically divergent Fusarium steppicola lineage.

In this study, DNA sequence data were used to characterize 290 Fusarium strains isolated during a survey of root-colonizing endophytic fungi of agricultural and nonagricultural plants in northern Kazakhstan. The Fusarium collection was screened for species identity using partial translation elongation factor 1-α (TEF1) gene sequences. Altogether, 16 different Fusarium species were identified, including eight known and four novel species, as well as the discovery of the phylogenetically divergent F. steppicola lineage. Isolates of the four putatively novel fusaria were further analyzed phylogenetically with a multilocus data set comprising partial sequences of TEF1, RNA polymerase II largest (RPB1) and second-largest (RPB2) subunits, and calmodulin (CaM) to assess their genealogical exclusivity. Based on the molecular phylogenetic and comprehensive morphological analyses, four new species are formally described herein: F. campestre, F. kazakhstanicum, F. rhizicola, and F. steppicola.

Dynamics of Carbendazim-Resistance Frequency of Pathogens Associated with the Epidemic of Fusarium Head Blight.

Carbendazim resistance was detected using 4,701 Fusarium graminearum species complex isolates collected from major wheat-producing regions in China from 2018 to 2020. A total of 348 carbendazim-resistant isolates were identified. The majority of carbendazim-resistant isolates were detected in Jiangsu and Anhui Provinces. In total, 227 and 88 isolates were obtained from each of the Jiangsu and Anhui Provinces, with a high resistance frequency of 41.12 and 20.56%, respectively. The predominant resistant isolates harboring point mutations were F167Y (79.31%), followed by E198Q (16.38%) and F200Y (4.31%). Compared with F. graminearum, F. asiaticum isolates were more likely to produce carbendazim resistance. In this study, we first detected carbendazim-resistant isolates in Hebei, Shaanxi, Sichuan, and Hunan Provinces. In Jiangsu, Anhui, and Zhejiang, the frequency of carbendazim-resistant isolates maintained a high level, resulting in stable carbendazim-resistant populations. We also found the dynamic of carbendazim-resistance frequency in most provinces showed similar trends to the epidemic of Fusarium Head Blight (FHB). Our results facilitate the understanding of the current situation of carbendazim resistance of FHB pathogens and will be helpful for fungicides selection in different wheat-producing areas in China.

Diversity regained: Precautionary approaches to COVID-19 as a phenomenon of the total environment.

As COVID-19 emerged as a phenomenon of the total environment, and despite the intertwined and complex relationships that make humanity an organic part of the Bio- and Geospheres, the majority of our responses to it have been corrective in character, with few or no consideration for unintended consequences which bring about further vulnerability to unanticipated global events. Tackling COVID-19 entails a systemic and precautionary approach to human-nature relations, which we frame as regaining diversity in the Geo-, Bio-, and Anthropospheres. Its implementation requires nothing short of an overhaul in the way we interact with and build knowledge from natural and social environments. Hence, we discuss the urgency of shifting from current to precautionary approaches to COVID-19 and look, through the lens of diversity, at the anticipated benefits in four systems crucially affecting and affected by the pandemic: health, land, knowledge and innovation. Our reflections offer a glimpse of the sort of changes needed, from pursuing planetary health and creating more harmonious forms of land use to providing a multi-level platform for other ways of knowing/understanding and turning innovation into a source of global public goods. These exemplary initiatives introduce and solidify systemic thinking in policymaking and move priorities from reaction-based strategies to precautionary frameworks.

Characterization of a G-quadruplex from hepatitis B virus and its stabilization by binding TMPyP4, BRACO19 and PhenDC3.

Specific guanine rich nucleic acid sequences can form non-canonical structures, like the four stranded G-quadruplex (GQ). We studied the GQ-forming sequence (named HepB) found in the genome of the hepatitis B virus. Fluorescence-, infrared- and CD-spectroscopy were used. HepB shows a hybrid form in presence of K+, but Na+, Li+, and Rb+ induce parallel structure. Higher concentrations of metal ions increase the unfolding temperature, which was explained by a short thermodynamic calculation. Temperature stability of the GQ structure was determined for all these ions. Na+ has stronger stabilizing effect on HepB than K+, which is highly unusual. The transition temperatures were 56.6, 53.8, 58.5 and 54.4 °C for Na+, K+, Li+, and Rb+ respectively. Binding constants for Na+ and K+ were 10.2 mM and 7.1 mM respectively. Study of three ligands designed in cancer research for GQ targeting (TMPyP4, BRACO19 and PhenDC3) showed unequivocally their binding to HepB. Binding was proven by the increased stability of the bound form. The stabilization was higher than 20 °C for TMPyP4 and PhenDC3, while it was considerably lower for BRACO19. These results might have medical importance in the fight against the hepatitis B virus.

Pressure Perturbation Studies of Noncanonical Viral Nucleic Acid Structures.

G-quadruplexes are noncanonical structures formed by guanine-rich sequences of the genome. They are found in crucial loci of the human genome, they take part in the regulation of important processes like cell proliferation and cell death. Much less is known about the subjects of this work, the viral G-quadruplexes. We have chosen three potentially G-quadruplex-forming sequences of hepatitis B. We measured the stability and the thermodynamic parameters of these quadruplexes. We also investigated the potential stabilization of these G-quadruplexes by binding a special ligand that was originally developed for cancer therapy. Fluorescence and infrared spectroscopic measurements were performed over wide temperature and pressure ranges. Our experiments indicate the small unfolding volume change of all three oligos. We found a difference between the unfolding of the 2-quartet and the 3-quartet G-quadruplexes. All three G-quadruplexes were stabilized by TMPyP4, which is a cationic porphyrin developed for stabilizing the human telomere.

What is the role of the nitrate reductase (euknr) gene in fungi that live in nitrate-free environments? A targeted gene knock-out study in Ampelomyces mycoparasites.

Mycoparasitic fungi can be utilized as biocontrol agents (BCAs) of many plant pathogens. Deciphering the molecular mechanisms of mycoparasitism may improve biocontrol efficiency. This work reports the first functional genetic studies in Ampelomyces, widespread mycoparasites and BCAs of powdery mildew fungi, and a molecular genetic toolbox for future works. The nitrate reductase (euknr) gene was targeted to reveal the biological function of nitrate assimilation in Ampelomyces. These mycoparasites live in an apparently nitrate-free environment, i.e. inside the hyphae of powdery mildew fungi that lack any nitrate uptake and assimilation system. Homologous recombination-based gene knock-out (KO) was applied to eliminate the euknr gene using Agrobacterium tumefaciens-mediated transformation. Efficient KO of euknr was confirmed by PCR, and visible phenotype caused by loss of euknr was detected on media with different nitrogen sources. Mycoparasitic ability was not affected by knocking out euknr as a tested transformant readily parasitized Blumeria graminis and Podosphaera xanthii colonies on barley and cucumber, respectively, and the rate of mycoparasitism did not differ from the wild type. These results indicate that euknr is not involved in mycoparasitism. Dissimilatory processes, involvement in nitric oxide metabolism, or other, yet undiscovered processes may explain why a functional euknr is maintained in Ampelomyces.

Negative volume changes of human G-quadruplexes at unfolding.

G-quadruplexes are tetrahelical structures. They are important targets for anti-cancer drugs, since they are situated at crucial positions within the genome. We studied the volumetric properties of the unfolding of three G-quadruplexes in the presence of potassium ion. The unfolding volume changes were determined using high-pressure fluorescence spectroscopy. The c-MYC, KIT, and VEGF sequences unfold with the transition volume of -17, -6 and -18 cm3/mol, respectively. The small magnitude of the unfolding volume of KIT could be explained by its unique structure and the lower amount of void volume. Since the cell interior is highly crowded, the available volume is restricted. Therefore the volumetric changes during the conformational transformations gain biological importance.

Revealing unfolding steps and volume changes of human telomeric i-motif DNA.

Cytosine rich DNA sequences can fold into a non-canonical four stranded intercalated i-motif structure. We investigated Htel-iM, an i-motif structure of the human telomeric DNA region that plays an important role in cell division, cancer diseases and aging. A high pressure up to 1 Gpa was applied to reveal the volumetric changes during unfolding. Thermal transitions at different pressures were followed by infrared and fluorescence spectroscopy. We demonstrated that Htel-iM unfolds in two steps. First the outer hydrogen bonds break in which C(2)[double bond, length as m-dash]O(2) and the N(4)H2 amino group is involved, subsequently the hydrogen bond involving N(3)+ becomes broken. Htel-iM was destabilized by pressure and unfolded with a negative volume change.

Relationship between oxidative stress and sexual coloration of lizards depends on thermal habitat.

Sexual signals serve as an honest indicator of individual quality, reflecting either developmental and/or maintenance costs. A possible underlying physiological mechanism is oxidative stress, which could mediate energy trade-offs between sexual signals and other quality traits. In ectotherms, thermal performance acts as a key indicator of individual quality and influence signal intensity. We investigated how oxidative state is reflected in visual signals of lizards from different thermal habitats. According to our hypothesis, efficient thermoregulation requires different strategies in different thermal environments. In a habitat with predictable temperature changes, animals are less exposed to suboptimal temperature ranges and selection will, therefore, be stronger on the maximum oxidative damage at optimal body temperature. Contrarily, in a habitat with rather stochastic thermal shifts, individuals are often constricted by suboptimal thermal conditions, and oxidative damage can be limiting on a wide temperature range. We used Iberolacerta cyreni and Psammodromus algirus inhabiting stochastic and predictable thermal environments respectively. We examined two aspects of oxidative stress: the level of reactive oxygen metabolites at the preferred temperature (maximal ROM) and the temperature range in which animals produce at least 80% of the maximum level of reactive oxygen metabolites (effective ROM range). In I. cyreni, we found that duller coloration was related to a wider effective ROM range, while expression of coloration in P. algirus was negatively correlated with the maximal ROM. Our results suggest that different thermal constraints affect different aspects of oxidative damage which can indicate individual quality and are, therefore, represented in sexual ornaments.

Environment-dependence of behavioural consistency in adult male European green lizards (Lacerta viridis).

Understanding the background mechanisms affecting the emergence and maintenance of consistent between-individual variation within population in single (animal personality) or across multiple (behavioural syndrome) behaviours has key importance. State-dependence theory suggests that behaviour is 'anchored' to individual state (e.g. body condition, gender, age) and behavioural consistency emerges through behavioural-state feedbacks. A number of relevant state variables are labile (e.g. body condition, physiological performance) and expected to be affected by short-term environmental change. Yet, whether short-term environmental shifts affect behavioural consistency during adulthood remains questionable. Here, by employing a full-factorial laboratory experiment, we explored if quantity of food (low vs. high) and time available for thermoregulation (3h vs. 10h per day) had an effect on activity and risk-taking of reproductive adult male European green lizards (Lacerta viridis). We focussed on different components of behavioural variation: (i) strength of behavioural consistency (repeatability for animal personality; between-individual correlation for behavioural syndrome), (ii) behavioural type (individual mean behaviour) and (iii) behavioural predictability (within-individual behavioural variation). Activity was repeatable in all treatments. Risk-taking was repeatable only in the low basking treatments. We found significant between-individual correlation only in the low food × long basking time group. The treatments did not affect behavioural type, but affected behavioural predictability. Activity predictability was higher in the short basking treatment, where it also decreased with size (≈ age). Risk-taking predictability in the short basking treatment increased with size under food limitation, but decreased when food supply was high. We conclude that short-term environmental change can alter various components of behavioural consistency. The effect could be detected in the presence/absence patterns of animal personality and behavioural syndrome and the level of individual behavioural predictability, but not in behavioural type.

Effects of energy and thermoregulation time on physiological state and sexual signal in a lizard.

Theory of sexual selection states that males often develop showy signals, which reduce their survival but increase their reproductive success. During mate choice, these conspicuous signals can be honest indicators of individual quality conveying information about the signaler's physiological state. Sexually selected signals are influenced by many environmental factors; however, whether signals and physiological state are affected together is rarely studied. The ultraviolet-blue throat color of male Lacerta viridis is an intra- and intersexually selected signal connected to blood parasite infection and influenced by environmental factors. The aim of this study was to experimentally investigate how ecologically relevant environmental factors affect color signal intensity and key physiological traits parallel. During the mating season, we exposed 40 adult male lizards infected with blood parasites to food and basking time treatments in a full factorial design. We measured color, amount of reactive oxygen metabolites (ROMs), hematocrit, immunocompetence, and blood parasite intensity before and after treatments. High basking time resulted in elevated immunocompetence coupled with increased ROMs. The high food treatment increased nuptial color brightness, but also increased ROMs and decreased immunocompetence. In summary, our study provides experimental evidence about environmentally induced parallel changes in an honest sexual signal and several quality-indicator physiological traits. We showed that available energy and time for high metabolism have independent and sometimes opposite effects on individual state.

Spatial variation in climate mediates gene flow across an island archipelago.

High levels of gene flow among partially isolated populations can overwhelm selection and limit local adaptation. This process, known as "gene swamping," can homogenize genetic diversity among populations and reduce the capacity of a species to withstand rapid environmental change. We studied brown anole lizards (Anolis sagrei) distributed across seven islands in The Bahamas. We used microsatellite markers to estimate gene flow among islands and then examined the correlation between thermal performance and island temperature. The thermal optimum for sprint performance was correlated with both mean and maximum island temperature, whereas performance breadth was not correlated with any measure of temperature variation. Gene flow between islands decreased as the difference between mean island temperatures increased, even when those islands were adjacent to one another. These data suggest that phenotypic variation is the result of either (1) local genetic adaptation with selection against immigrants maintaining variation in the thermal optimum, (2) irreversible forms of adaptive plasticity such that immigrants have reduced fitness, or (3) an interaction between fixed genetic differences and plasticity. In general, the patterns of gene flow we observed suggest that local thermal environments represent important ecological filters that can mediate gene flow on relatively fine geographic scales.

Occurrence of Fusarium verticillioides and Fusarium musae on banana fruits marketed in Hungary.

Fusarium strains were isolated from rotten banana fruit imported into Hungary from some African and some Neotropical countries. The strains were identified using morphological features, 2-benzoxazolinone tolerance, translation elongation factor (EF-1α) sequences and inter simple sequence repeat (ISSR) analysis. All strains from Africa proved to be F. verticillioides whereas the strains from the Neotropics are Fusarium musae. According to the PCR proof and the fumonisin toxin measurement F. musae strains cannot produce any fumonisins (FB1-4).

European green lizard (Lacerta viridis) personalities: Linking behavioural types to ecologically relevant traits at different ontogenetic stages.

Consistent individual differences within (animal personality) and across (behavioural syndrome) behaviours became well recognized during the past decade. Nevertheless, our knowledge about the evolutionary and developmental mechanisms behind the phenomena is still incomplete. Here, we explored if risk-taking and exploration were consistent and linked to different ecologically relevant traits in wild-caught adult male European green lizards (Lacerta viridis) and in their 2-3 weeks old laboratory-reared offspring. Both adults and juveniles displayed animal personality, consistency being higher in juveniles. We found correlation between risk-taking and exploration (suggestive of a behavioural syndrome) only in adults. Juveniles were more explorative than adults. Large or ectoparasite-free adult males were more explorative than small or parasitized males. Juvenile females tended to be more risk-taking than males. Behaviour of fathers and their offspring did not correlate. We conclude that European green lizards show high behavioural consistency and age is an important determinant of its strength and links to traits likely affecting fitness.

Negative correlation between nuptial throat colour and blood parasite load in male European green lizards supports the Hamilton-Zuk hypothesis.

During female mate choice, conspicuous male sexual signals are used to infer male quality and choose the best sire for the offspring. The theory of parasite-mediated sexual selection (Hamilton-Zuk hypothesis) presumes that parasite infection can influence the elaboration of sexual signals: resistant individuals can invest more energy into signal expression and thus advertise their individual quality through signal intensity. By preferring these males, females can provide resistance genes for their offspring. Previous research showed that nuptial throat colour of male European green lizard, Lacerta viridis, plays a role in both inter- and intrasexual selections as a condition-dependent multiple signalling system. The aim of this study was to test the predictions of the Hamilton-Zuk hypothesis on male European green lizards. By blood sampling 30 adult males during the reproductive season, we found members of the Haemogregarinidae family in all but one individual (prevalence = 96%). The infection intensity showed strong negative correlation with the throat and belly colour brightness in line with the predictions of the Hamilton-Zuk hypothesis. In addition, we found other correlations between infection intensity and other fitness-related traits, suggesting that parasite load has a remarkable effect on individual fitness. This study shows that throat patch colour of the European green lizards not only is a multiple signalling system but also possibly acts as an honest sexual signal of health state in accordance with the Hamilton-Zuk hypothesis.

Temperature, but not available energy, affects the expression of a sexually selected ultraviolet (UV) colour trait in male European green lizards.

BACKGROUND: Colour signals are widely used in intraspecific communication and often linked to individual fitness. The development of some pigment-based (e.g. carotenoids) colours is often environment-dependent and costly for the signaller, however, for structural colours (e.g. ultraviolet [UV]) this topic is poorly understood, especially in terrestrial ectothermic vertebrates. METHODOLOGY/PRINCIPAL FINDINGS: In a factorial experiment, we studied how available energy and time at elevated body temperature affects the annual expression of the nuptial throat colour patch in male European green lizards (Lacerta viridis) after hibernation and before mating season. In this species, there is a female preference for males with high throat UV reflectance, and males with high UV reflectance are more likely to win fights. We found that (i) while food shortage decreased lizards' body condition, it did not affect colour development, and (ii) the available time for maintaining high body temperature affected the development of UV colour without affecting body condition or other colour traits. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that the expression of a sexually selected structural colour signal depends on the time at elevated body temperature affecting physiological performance but not on available energy gained from food per se in an ectothermic vertebrate. We suggest that the effect of high ambient temperature on UV colour in male L. viridis makes it an honest signal, because success in acquiring thermally favourable territories and/or effective behavioural thermoregulation can both be linked to individual quality.