Sampling Optimization and Crop Interface Effects on Lygus lineolaris Populations in Southeastern USA Cotton

Tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae), is an economically damaging pest in cotton production systems across the southern United States. We systematically scouted 120 commercial cotton fields across five southeastern states during susceptible growth stages in 2019 and 2020 to investigate sampling optimization and the effect of interface crop and landscape composition on L. lineolaris abundance. Variance component analysis determined field and within-field spatial scales, compared with agricultural district and state, accounted for more variation in L. lineolaris density using sweep net and drop cloth sampling. This result highlights the importance of field-level scouting efforts. Using within-field samples, a fixed-precision sampling plan determined 8 and 23 sampling units were needed to determine L. lineolaris population estimates with 0.25 precision for sweep net (100 sweeps per unit) and drop cloth (1.5 row-m per unit) sampling, respectively. A spatial Bayesian hierarchical model was developed to determine local landscape (<0.5 km from field edges) effects on L. lineolaris in cotton. The proportion of agricultural area and double-crop wheat and soybeans were positively associated with L. lineolaris density, and fields with more contiguous cotton areas negatively predicted L. lineolaris populations. These results will improve L. lineolaris monitoring programs and treatment management decisions in southeastern USA cotton.

Identification of Pathogenic Fusarium spp. Responsible for Root Rot of Angelica sinensis and Characterization of their Biological Enemies in Dingxi, China

Root rot is a serious disease in plantations of A. sinensis, severely affecting yield and quality and threatening sustainable production. Fusarium isolates (n=32) were obtained from field samples of root rot tissue, leaves and infected soil. Isolates were identified by comparing the sequences of their internal transcribed spacer (ITS) region and translation elongation factor 1-ɑ (TEF-1ɑ) to sequences of known species in the NCBI-database. These Fusarium isolates include F. tricinctum (43.75%), F. equiseti (31.25%), F. solani (9.37%), F. oxysporum (6.25%), F. acuminatum (6.25%), and F. incarnatum (3.12%). For pathogenicity testing under greenhouse conditions, seven isolates were selected based on a phylogenetic analysis, including four strains of F. tricinctum and one strain each of F. solani, F. oxysporum, and F. acuminatum. The seven isolates were all pathogenic but differed in their ability to infect: the four F. tricinctum strains were capable pathogens causing root rot in A. sinensis at 100% incidence and the highly aggressive. Furthermore, the symptoms of root rot induced by those seven isolates were consistent with typical root rot cases in the field, but their disease severity varied. Observed histopathological preparations of F. tricinctum-infected seedlings and tissue-slides results showed this fungal species can penetrate epidermal cells and colonize the cortical cells where it induces necrosis and severe plasmolysis. Plate confrontation experiments showed that isolated rhizosphere bacteria inhibited the Fusarium pathogens that cause root rot in A. sinensis. Our results provide timely information for informing the use of biocontrol agents for suppression of root rot disease.

Improved detection of Little cherry virus-2 using a hydrolysis probe to manage the Pacific Northwest little cherry disease epidemic.

Little cherry virus-2 (LChV-2) is a viral pathogen that is reaching epidemic levels in Washington state. This virus is insect-vectored and has significant impacts on sweet cherry production. To aid growers in making informed management decisions we sought to develop a diagnostic assay to better detect isolates of LChV-2 currently found in Washington, allowing for more accurate estimations of disease occurrence. This study showed that there were two distinct genotypes of LChV-2 present in Washington state. This information was used to develop an up-to-date reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) assay, which was then optimized, validated, and compared to four previously published assays using a panel of field samples. This comparison demonstrated that the newly developed assay provided greater sensitivity, accurately detecting less than 10 copies per reaction and could detect both LChV-2 genotypes. Finally, we examined the effect of potential inhibitors in various tissue types from cherry, finding that young leaf tissue affected sensitivity of detection less than root tissues.

HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in &lt; 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

Population-level genome-wide STR discovery and validation for population structure and genetic diversity assessment of Plasmodium species

Short tandem repeats (STRs) are highly informative genetic markers that have been used extensively in population genetics analysis. They are an important source of genetic diversity and can also have functional impact. Despite the availability of bioinformatic methods that permit large-scale genome-wide genotyping of STRs from whole genome sequencing data, they have not previously been applied to sequencing data from large collections of malaria parasite field samples. Here, we have genotyped STRs using HipSTR in more than 3,000 Plasmodium falciparum and 174 Plasmodium vivax published whole-genome sequence data from samples collected across the globe. High levels of noise and variability in the resultant callset necessitated the development of a novel method for quality control of STR genotype calls. A set of high-quality STR loci (6,768 from P. falciparum and 3,496 from P. vivax) were used to study Plasmodium genetic diversity, population structures and genomic signatures of selection and these were compared to genome-wide single nucleotide polymorphism (SNP) genotyping data. In addition, the genome-wide information about genetic variation and other characteristics of STRs in P. falciparum and P. vivax have been available in an interactive web-based R Shiny application PlasmoSTR (https://github.com/bahlolab/PlasmoSTR).

Experimental Study on the Reliability of Scaling Down Techniques Used in Direct Shear Tests to Determine the Shear Strength of Rockfill and Waste Rocks

The determination of shear strength parameters for coarse granular materials such as rockfill and waste rocks is challenging due to their oversized particles and the minimum required ratio of 10 between the specimen width (W) and the maximum particle size (dmax) of tested samples for direct shear tests. To overcome this problem, a common practice is to prepare test samples by excluding the oversized particles. This method is called the scalping scaling down technique. Making further modifications on scalped samples to achieve a specific particle size distribution curve (PSDC) leads to other scaling down techniques. Until now, the parallel scaling down technique has been the most popular and most commonly applied, generally because it produces a PSDC parallel and similar to that of field material. Recently, a critical literature review performed by the authors revealed that the methodology used by previous researchers to validate or invalidate the scaling down techniques in estimating the shear strength of field materials is inappropriate. The validity of scaling down techniques remains unknown. In addition, the minimum required W/dmax ratio of 10, stipulated in ASTM D3080/D3080M-11 for direct shear tests, is not large enough to eliminate the specimen size effect (SSE). The authors’ recent experimental study showed that a minimum W/dmax ratio of 60 is necessary to avoid any SSE in direct shear tests. In this study, a series of direct shear tests were performed on samples with different dmax values, prepared by applying scalping and parallel scaling down techniques. All tested specimens had a W/dmax ratio equal to or larger than 60. The test results of the scaled down samples with dmax values smaller than those of field samples were used to establish a predictive equation between the effective internal friction angle (hereafter named “friction angle”) and dmax, which was then used to predict the friction angles of the field samples. Comparisons between the measured and predicted friction angles of field samples demonstrated that the equations based on scalping scaling down technique correctly predicted the friction angles of field samples, whereas the equations based on parallel scaling down technique failed to correctly predict the friction angles of field samples. The scalping down technique has been validated, whereas the parallel scaling down technique has been invalidated by the experimental results presented in this study.

Identification and Characterization of P0 Protein as a Vaccine Candidate Against Babesia divergens, Blood Parasite of Veterinary and Zoonotic Importance

The molecular identification and antigenic characterization of P0 protein in Babesia divergens, a blood parasite of veterinary and zoonotic importance, were carried out in this study for use in developing subunit vaccines against B. divergens infection. Recombinant protein encoding P0 (BdP0) was developed in Escherichia coli, and its antiserum was generated in mice for further molecular characterization. Anti-rBdP0 serum had a specific interaction with the corresponding legitimate B. divergens protein, as confirmed by Western blotting and indirect fluorescent antibody tests. ELISA was used to assess the immunogenicity of BdP0 in a group of 68 bovine field samples, and significant immunological reactivity was found in 19 and 20 positive samples of rBdp0 and B. divergens lysate, respectively. The in vitro growth of B. divergens cultures treated with anti-rBdP0 serum was significantly inhibited (p &lt; 0.05). Furthermore, after 6 h of incubation with 2 mg/ml anti-rBdP0 serum, the ability of pre-incubated free merozoites to invade bovine erythrocytes was reduced by 59.88%. The obtained data suggest the possible use of rBdP0 as diagnostic antigen and may serve as a vaccine candidate against babesiosis caused by B. divergens either in animal or human.

First report of Alternaria alternata causing the golden spot in xoconostle (Opuntia matudae) in Hidalgo, México

The species<em> Opuntia ficus-indica</em> is affected by pests and diseases, one of the most important of these being golden spot. The latter has been reported in various countries, including Mexico. Symptoms similar to those of golden spot in <em>O. ficus-indica</em> have been observed in the xoconostle crop (<em>Opuntia matudae</em> Scheinvar, cv. Rosa). The objective of this study was to isolate and identify the causative agent that causes the symptoms, which resembled those of the disease described as golden spot in <em>O. ficus-indica</em>. Simple random sampling in the field (10 plots) was carried out on xoconostle plants that presented the described symptoms. Slices of diseased plant tissue were cut and placed in Petri plates with PDA medium. Compliance with Koch’s postulates showed that the isolated (one aislated) of <em>Alternaria</em> sp. obtained from the field samples colonized the cladodes after inoculations in the greenhouse. Through taxonomic keys, <em>Alternaria</em> sp. was identified as the causative microorganism. Molecular characterization of the isolated <em>Alternaria</em> sp. was identified as <em>Alternaria</em> <em>alternata</em>. This is the first report worldwide of <em>Alternaria alternata</em> as the causal agent of golden spot in a xoconostle crop.

Development of a Recombinase Polymerase Amplification Fluorescence Assay for the Detection of Canine Adenovirus 2

Canine adenovirus type 2 (CAdV-2) is often found in co-infections with other pathogens causing canine infectious respiratory disease (CIRD). Rapid, efficient, and convenient pathogen detection is the best approach for early confirmatory diagnosis. In this study, we developed and evaluated a rapid real-time recombinase polymerase amplification (RPA) assay for detection of canine adenovirus 2 (CAV), which can detect CAV within 15 min at 39°C. The detection limit that assay was 214 copies/μl DNA molecules per reaction. The specificity was indicated by a lack of cross-reaction with canine distemper virus (CDV), canine coronavirus (CCV), and canine parvovirus (CPV). Field and clinical applicability of this assay were evaluated using 86 field samples. The coincidence rate of the detection results for clinical samples between CAV-RPA and qPCR was 97.7%. In summary, the real-time CAV-RPA analysis provides an efficient, rapid and sensitive detection method for CAV.