The Role of Soil Abundance of TxtAB in Potato Common Scab Disease Severity.

Common scab is an economically costly soilborne disease of potato endemic in many potato-growing regions. The disease is caused by species of Streptomyces bacteria that produce the phytotoxin thaxtomin A. The primary disease management tool available to growers is planting resistant cultivars, but no cultivar is fully resistant to common scab, and partially resistant cultivars are often not the preferred choice of growers because of agronomic or market considerations. Therefore, growers would benefit from knowledge of the presence and severity of common scab infestations in field soils to make informed planting decisions. We implemented a quantitative PCR diagnostic assay to enable field detection and quantification of all strains of Streptomyces that cause common scab in the United States through amplification of thaxtomin A biosynthetic genes. Greenhouse trials confirmed that pathogen abundance was highly correlated with disease severity for five distinct phytopathogenic Streptomyces species, although the degree of disease severity was dependent on the pathogen species. Correlations between the abundance of the thaxtomin biosynthetic genes from field soil with disease on tubers at field sites across four U.S. states and across 2 years were not as strong as correlations observed in greenhouse assays. We also developed an effective droplet digital PCR diagnostic assay that also has potential for field quantification of thaxtomin biosynthetic genes. Further improvement of the PCR assays and added modeling of other environmental factors that impact disease outcome, such as soil composition, can aid growers in making informed planting decisions.

An In-Depth Evaluation of Powdery Mildew Hosts Reveals One of the World's Most Common and Widespread Groups of Fungal Plant Pathogens.

Powdery mildews are highly destructive fungal plant pathogens that have a significant economic impact on both agricultural and ecological systems worldwide. The intricate relationship between powdery mildews and their host plants has led to cospeciation. In this study, we conducted an extensive evaluation of powdery mildew hosts to provide an updated understanding of the host ranges and distributions of these fungi. The "United States National Fungus Collections Fungus-Host Dataset" is the primary source of information for our analyses. The analysis of the dataset demonstrated the worldwide prevalence of powdery mildews; the data contained over 72,000 reports of powdery mildews, representing ∼8.7% of all host-fungal records. We have updated the taxonomy and nomenclature of powdery mildews. In total, powdery mildews infect ∼10,125 host taxa belonging to 205 families of flowering plants, which accounts for 1,970 genera in 200 countries across six continents. Furthermore, we estimate that powdery mildews infect approximately 2.9% of described angiosperm species. Our study underscores the need for regular updates on powdery mildew host information due to the continuously evolving taxonomy and the discovery of new host taxa. Since 1986, we estimate an additional 1,866 host taxa, 353 genera, and 36 families have been reported. Additionally, the identification of powdery mildew hosts provides valuable insights into the coevolutionary dynamics between the fungi and their plant hosts. Overall, this updated list provides valuable insights into the taxonomy and geographic distribution of powdery mildew species, which builds upon the previous work of Amano in 1986. Discerning the geographic spread and host range of economically significant plant pathogens is vital for biosecurity measures and identifying the origins and expansion of potentially harmful pathogens.

Extensive intragenomic variation in the internal transcribed spacer region of fungi.

Fungi are among the most biodiverse organisms in the world. Accurate species identification is imperative for studies on fungal ecology and evolution. The internal transcribed spacer (ITS) rDNA region has been widely accepted as the universal barcode for fungi. However, several recent studies have uncovered intragenomic sequence variation within the ITS in multiple fungal species. Here, we mined the genome of 2414 fungal species to determine the prevalence of intragenomic variation and found that the genomes of 641 species, about one-quarter of the 2414 species examined, contained multiple ITS copies. Of those 641 species, 419 (∼65%) contained variation among copies revealing that intragenomic variation is common in fungi. We proceeded to show how these copies could result in the erroneous description of hundreds of fungal species and skew studies evaluating environmental DNA (eDNA) especially when making diversity estimates. Additionally, many genomes were found to be contaminated, especially those of unculturable fungi.

Evaluating the Effectiveness of Interventions to Improve the Follow-up Rate for Children With Visual Disabilities in an Eye Hospital in Nepal: Nonrandomized Study.

BACKGROUND: Monitoring ocular morbidity among pediatric patients requires regular follow-up visits. We found that the follow-up rate was poor among children in our setting. Therefore, we intended to assess the effectiveness of 2 interventions-(1) counseling and (2) SMS text messaging and phone calls-to improve the follow-up rates. OBJECTIVE: This study aimed to evaluate the effectiveness of 2 interventions, counseling and SMS and phone calls group, as well as a routine standard care for improving the follow-up rate of pediatric patients. METHODS: A Nonrandomized, quasiexperimental design was used. Children (aged 0-16 years) with ocular conditions requiring at least 3 follow-up visits during the study period were included. A total of 264 participants were equally allocated to the 3 intervention groups of (1) counseling, (2) SMS and phone calls, and (3) routine standard care group. A 20-minute counseling session by a trained counselor with the provision of disease-specific leaflets were given to those in the counseling group. For the second intervention group, parents of children received an SMS text 3 days before and a phone call 1 day before their scheduled follow-up visits. Participants allocated for the routine standard care group were provided with the existing services with no additional counseling and reminders. Participants attending 3 follow-ups within 2 days of the scheduled visit date were considered compliant. The difference in and among the proportion of participants completing all 3 follow-up visits in each group was assessed. RESULTS: The demographic characteristics of the participants were similar across the study groups. Only 3% (8/264) of participants completed all 3 follow-up visits, but overall compliance with the follow-up, as defined by the investigators, was found to be only 0.76% (2/264). There was no statistically significant difference in the proportion of follow-up between the intervention groups. However, the proportion of participants attending the first and second follow-ups, as well as the overall total number of follow-ups, was more in the SMS and phone-call group followed by the counseling group. CONCLUSIONS: We did not find any evidence on the effectiveness of our interventions to improve the follow-up rate. The primary reason could be that this study was conducted during the COVID-19 pandemic. It could also be possible that the intensity of the interventions may have influenced the outcomes. A rigorously designed study during the absence of any lockdown restrictions is warranted to evaluate intervention effectiveness. The study also provides useful insights and highlights the importance of designing and systematically developing interventions for improving the follow-up rate and ensuring a continuum of care to children with visual disabilities in Nepal and similar contexts. TRIAL REGISTRATION: ClinicalTrials.gov NCT04837534; https://clinicaltrials.gov/ct2/show/NCT04837534. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/31578.

Improving the Follow-up Rate for Pediatric Patients (0-16 years) of an Eye Hospital in Nepal: Protocol for a Public Health Intervention Study.

BACKGROUND: The follow-up of pediatric patients ensures regular ocular morbidity monitoring and better treatment outcome. Hiralal Santudevi Pradhan Institute of Ophthalmic Science (Bharatpur Eye Hospital [BEH]) noticed that the follow-up rate was only 22% among its pediatric patients. Several factors like lack of awareness and forgetfulness among patients may contribute to a lower number of follow-up visits. Therefore, BEH decided to find if counseling and reminders through SMS text messaging and phone calls would improve the follow-up rates. OBJECTIVE: This study aims to evaluate the impact of interventions like counseling and reminder SMS text messaging and phone calls in improving the follow-up rate of pediatric patients. METHODS: This is a public health intervention study being conducted using quantitative analysis. All children (0-16 years) with ocular conditions requiring at least 3 follow-up visits in the study period will be included. In all, 264 participants will be allocated to 3 groups: routine standard care, counseling, and reminders with SMS text messaging and phone calls. In counseling, patients will take part in 20-minute counseling sessions with trained counselors at each visit, and information leaflets will be provided to them. In the reminder SMS text messaging and phone call group, patients will receive an SMS text message 3 days prior and a phone call 1 day prior to their scheduled visits. Patients attending within 2 days of the scheduled date will be considered compliant to follow-up. The proportion of patients completing all the follow-up visits in each group will be assessed. Informed consent will be taken from parents and children. Univariate and multivariate analyses will be conducted. RESULTS: The ethical approval for this study has been obtained from the Ethical Review Board (ERB) of Nepal Health Research Council (ERB protocol registration #761/2020 P). The data collection was initiated on January, 24, 2021, but due to the COVID-19 pandemic, as of September 2021, we have only been able to enroll 154 of the planned 264 participants (58.3% of the sample size). CONCLUSIONS: This study will reliably document not only the factors associated with follow-up rate through an intervention package (counseling and reminders through SMS text messaging and phone calls) but also the cost effectiveness of the intervention package, which can be applied in all the departments of the hospital. TRIAL REGISTRATION: ClinicalTrials.gov NCT04837534; https://clinicaltrials.gov/ct2/show/NCT04837534. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/31578.

Factors Affecting the Infection of Sweet Cherry (Prunus avium) Fruit by Podosphaera cerasi.

Powdery mildew caused by Podosphaera cerasi is the most important fungal disease of sweet cherries in the Pacific Northwest of the United States. In this study, several factors related to disease epidemiology were evaluated. The experiments were conducted to investigate flower susceptibility to P. cerasi infection by in planta and in vitro inoculation. The susceptibility of fruit at various developmental stages was investigated using defined concentrations of P. cerasi conidia. Furthermore, the threshold of conidial concentration required for fruit infection was determined. The pathogen activity during full bloom was limited and not related to fruit disease incidence and severity at harvest. Foliar infections always preceded fruit infections by an average of 42 days during the 3 years of the study. The onset of fruit infection followed, on average, 66 days after full bloom and appeared simultaneously on all susceptible cherry cultivars in the research orchard. Disease symptoms were only observed on fruit in Biologische Bundesanstalt, Bundessortenamt, and Chemical Industry scale 8 (maturity) in all cultivars examined. During this stage, a concentration of 500 conidia/ml was sufficient to cause fruit infection at harvest. Interaction between the inoculation dates and conidial concentration revealed a dependency of disease development on the host stage at the time of inoculation; the younger the fruit, the more conidia are needed to cause disease at harvest. Molecular studies showed a rapid increase in conidia viability at the transition from asymptomatic to the symptomatic disease of fruit. No evidence of ontogenic resistance of fruit to powdery mildew infection was observed.

Development and Application of a Real-Time Reverse-Transcription PCR and Droplet Digital PCR Assays for the Direct Detection of Potato mop top virus in Soil.

Potato mop top virus (PMTV) is a continuing threat to potato production throughout the world. It has the potential to persist in the soil for long periods in the sporosori of its vector Spongospora subterranea f. sp. subterranea, which is as an important source for PMTV infection and dissemination. In this study, we used real-time quantitative reverse-transcription PCR (qRT-PCR) and reverse-transcription droplet digital PCR (RT-ddPCR) assays of the total RNA extracted directly from the soil to develop a simple, fast, and sensitive method to detect PMTV in soil samples using a specific primer with high efficiency despite a minimal amount of viral RNA. The designed primers are resilient in the presence of various PCR inhibitors in the soil when RNA is extracted. Both assays detected PMTV in all soil types used and supported the detection of <10 PMTV copies µl-1 in the RNA sample. With qRT-PCR, detection was linear, with amplification efficiencies ranging from 93.3 to 105.3% for silt loam, loamy sand, sand, and sandy loam in various experiments with R2 > 0.99. Furthermore, the RT-ddPCR assay also demonstrated a high degree of linearity (R2 > 0.99 and P < 0.0001) with the RNA extracted from the soil samples representing different textures and physiochemical characteristics that were artificially spiked with infested S. subterranea f. sp. subterranea sporosori. Additionally, both assays successfully detected PMTV in different types of naturally infested soil with PMTV carrying S. subterranea f. sp. subterranea sporosori levels ranging from 6.2 × 102 g-1 to 1.2 × 106 g-1 in soils with pH ranging from 4.9 to 7.5 and organic matter ranging from 0.9 to 5.1%, demonstrating the potential to detect PMTV in a wide variety of soils. To our knowledge, this is the first report of the development of real-time PCR and ddPCR methods for the direct detection of a soilborne virus in soil.

Phylogeny and taxonomy of Podosphaera cerasi, sp. nov., and Podosphaera prunicola sensu lato.

Powdery mildew of Prunus spp. is a significant disease in most cherry growing regions of Washington, USA. Powdery mildews on Prunus virginiana and Pr. avium were previously assigned to Podosphaera clandestina s. lat. (= Po. oxyacanthae) or Po. prunicola. In this report, we confirm the presence of two distinct Podosphaera species on these hosts. Phylogenetic analyses of nuc rDNA sequences from the internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS) and 28S subunit confirmed the presence of two distinct species. A morphological comparison with type material of Po. prunicola and additional collections demonstrated that the powdery mildew on Pr. virginiana (including var. demissa and var. melanocarpa) is in fact Po. prunicola. The powdery mildew on Pr. avium is genetically, morphologically, and biologically distinct from Po. prunicola and is described here as the new species Po. cerasi. Cross-inoculation experiments confirmed that these two species are host specific. Podosphaera prunicola was unable to colonize Pr. avium, whereas Po. cerasi was unable to colonize Pr. virginiana. Morphological reexamination of numerous specimens identified as Po. prunicola on a broad range of Prunus species suggests that Po. prunicola is probably confined to species in Prunus subgen.Padus (= Prunus subgen. Cerasus sect. Laurocerasus, including sect. Padus), with Pr. virginiana as the principal host. Podosphaera cerasi occurs on hosts in Prunus subgen. Cerasus, and our work confirms a newly described species of powdery mildew on Pr. avium. This work also includes the first documented and genetically proven European record of Po. prunicola on Pr. serotina and its widespread occurrence in the United States.

Characterization of human translesion DNA synthesis across a UV-induced DNA lesion.

Translesion DNA synthesis (TLS) during S-phase uses specialized TLS DNA polymerases to replicate a DNA lesion, allowing stringent DNA synthesis to resume beyond the offending damage. Human TLS involves the conjugation of ubiquitin to PCNA clamps encircling damaged DNA and the role of this post-translational modification is under scrutiny. A widely-accepted model purports that ubiquitinated PCNA recruits TLS polymerases such as pol η to sites of DNA damage where they may also displace a blocked replicative polymerase. We provide extensive quantitative evidence that the binding of pol η to PCNA and the ensuing TLS are both independent of PCNA ubiquitination. Rather, the unique properties of pols η and δ are attuned to promote an efficient and passive exchange of polymerases during TLS on the lagging strand.

Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis.

In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion.

Electrochemical Impedance Spectroscopy Study of Concanavalin A Binding to Self-Assembled Monolayers of Mannosides on Gold Wire Electrodes.

The interactions of the lectin Concanavalin A (Con A) with self-assembled monolayers (SAMs) of thiolated mono-, di-, and tri-mannosides were studied on the surface of gold wires using electrochemical impedance spectroscopy (EIS). The SAMs of mannosides were prepared either pure or along with thiolated triethylene glycol (TEG) at different molar ratios (1:1, 1:2, 1:4, 1:9, and 1:19) to better understand and optimize the interaction conditions. The charge-transfer resistance of the [Fe(CN)6]3-/4- redox probe was compared before and after the interaction at different concentrations of Con A to determine the equilibrium dissociation constant (Kd) and limit of detection (LOD). Values of Kd were found in the nanomolar range showing multivalent interactions between mannosides and Con A, and LOD was found ranging from 4-13 nM depending on the type of mannoside SAM used. Analysis using the Hill equation suggests negative cooperativity in the binding behavior. Peanut agglutinin was used as a negative control, and cyclic voltammetry was used to further support the experiments. We have found that neither the pure nor the widely dispersed monolayers of mannosides provide the conditions for optimal binding of Con A. The binding of Con A to these SAMs is sensitive to the molar ratio of the mannoside used to prepare the SAM and to the structure of the mannoside. A simple cleaning method has also been shown to regenerate the used gold wire electrodes. The results from these experiments contribute to the development of simple, cheap, selective, and sensitive EIS-based bioassays, especially for lectin-carbohydrate interactions.

Square-wave voltammetry assays for glycoproteins on nanoporous gold.

Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A - ALP (or soybean agglutinin - ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A-ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL-1 BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 µM and 286 µM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay.

Electrochemical characterization of globotriose-containing self-assembled monolayers on nanoporous gold and their binding of soybean agglutinin.

Self-assembled monolayers (SAMs) of α-D-Gal-(1→4)-ß-D-Gal-(1→4)-ß-D-Glc-mercaptooctane (globotriose, Gb3-C8-SH) were prepared both as single-component SAMs and as mixed SAMs with either octanethiol (OCT) or 8-mercapto-3,6-dioxaoctanol (HO-PEG2-SH), on flat gold and on nanoporous gold (NPG) electrodes. The binding of soybean agglutinin (SBA) to the globotriose (Gb3) unit in the SAMs was then studied using electrochemical impedance spectroscopy (EIS), which is a label free method found to be quite sensitive to SAM composition and to the differences in SAM structure on NPG versus on flat Au. The affinity of SBA to the mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG is found to be greater on NPG than on flat gold, and indicates a potential advantage for NPG as a substrate. The SAMs of HO-PEG2-SH were found to resist protein adsorption on either NPG or flat gold. The non-specific adsorption of SBA to OCT SAMs on flat Au was observed in EIS by the increase in charge transfer resistance; whereas, the increase seen on the NPG surface was smaller, and suggests that EIS measurements on NPG are less affected by non-specific protein adsorption. Atomic force microscopy (AFM) images of the SBA binding to mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG showed a greater number of proteins on top of the OCT containing SAMs.

Comparative Study of the Binding of Concanavalin A to Self-Assembled Monolayers Containing a Thiolated α-Mannoside on Flat Gold and on Nanoporous Gold.

We have prepared SAMs containing 8-mercaptooctyl α-D-mannopyranoside, either as a single component or in mixed SAMs with n-octanethiol on flat gold surfaces and on nanoporous gold. Electrochemical impedance spectroscopy showed that the mixed SAMs on flat gold surfaces showed the highest Con A binding near 1:9 solution molar ratio of thiolatedα-mannoside to n-octanethiol whereas those on NPG showed the highest response at 1:19 solution molar ratio of thiolated α-mannoside to n-octanethiol. Atomic force microscopy was employed to image the monolayers, and also to image the bound Con A protein.

Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen.

Nanoporous gold (NPG) was utilized as a support for immobilizing alkaline phosphatase (ALP) conjugated to monoclonal antibodies against either prostate specific antigen (PSA) or carcinoembryonic antigen (CEA). The antibody-ALP conjugates were coupled to self-assembled monolayers of lipoic acid and used in direct kinetic assays. Using the enzyme substrate p-aminophenylphosphate, the product p-aminophenol was detected by its oxidation near 0.1 V (vs. Ag|AgCl) using square wave voltammetry. The difference in peak current arising from oxidation of p-aminophenol before and after incubation with biomarker increased with biomarker concentration. The response to these two biomarkers was linear up to 10 ng mL-1 for CEA and up to 30 ng mL-1 for PSA. The effect of interference on the PSA assay was studied using bovine serum albumin (BSA) as a model albumin protein. The effect of interference from a serum matrix was examined for the PSA assay using newborn calf serum. A competitive version of the immunoassay using antigen immobilized onto the NPG surface was highly sensitive at lower antigen concentration. Estimates of the surface coverage of the antibody-ALP conjugates on the NPG surface are presented.