Positive associations matter: Microbial relationships drive tick microbiome composition.

Untangling how factors such as environment, host, associations among bacterial species and dispersal predict microbial composition is a fundamental challenge. In this study, we use complementary machine-learning approaches to quantify the relative role of these factors in shaping microbiome variation of the blacklegged tick Ixodes scapularis. I. scapularis is the most important vector for Borrelia burgdorferi (the causative agent for Lyme disease) in the U.S. as well as a range of other important zoonotic pathogens. Yet the relative role of the interactions between pathogens and symbionts compared to other ecological forces is unknown. We found that positive associations between microbes where the occurrence of one microbe increases the probability of observing another, including between both pathogens and symbionts, was by far the most important factor shaping the tick microbiome. Microclimate and host factors played an important role for a subset of the tick microbiome including Borrelia (Borreliella) and Ralstonia, but for the majority of microbes, environmental and host variables were poor predictors at a regional scale. This study provides new hypotheses on how pathogens and symbionts might interact within tick species, as well as valuable predictions for how some taxa may respond to changing climate.

Multigenic hairpin transgenes in tomato confer resistance to multiple orthotospoviruses including Sw-5 resistance-breaking tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) and related thrips-borne orthotospoviruses are a threat to food and ornamental crops. Orthotospoviruses have the capacity for rapid genetic change by genome segment reassortment and mutation. Genetic resistance is one of the most effective strategies for managing orthotospoviruses, but there are multiple examples of resistance gene breakdown. Our goal was to develop effective multigenic, broad-spectrum resistance to TSWV and other orthotospoviruses. The most conserved sequences for each open reading frame (ORF) of the TSWV genome were identified and comparison to other orthotospoviruses revealed sequence conservation within virus clades and some overlapped with domains with well-documented biological functions. We made six hairpin constructs, each of which incorporated sequences matching portions of all five ORFs. Tomato plants expressing the hairpin transgene were challenged with TSWV by thrips and leaf-rub inoculation and four constructs provided strong protection against TSWV in foliage and fruit. To determine if the hairpin constructs provided protection against other emerging orthotospoviruses, we challenged the plants with tomato chlorotic spot virus and resistance-breaking TSWV (RB-TSWV) and found that the same constructs also provided resistance to these related viruses. Antiviral hairpin constructs are an effective way to protect plants from multiple orthotospoviruses and are an important strategy in the fight against RB-TSWV and emerging viruses. Targeting of all five viral ORFs is expected to increase the durability of resistance and combining them with other resistance genes could further extend the utility of this disease control strategy.

Athlete External Load Measures Across a Competitive Season in High School Basketball.

ABSTRACT: Askow, AT, Jennings, W, Jagim, AR, Fields, JB, Beaudoin, RG, Sanchez, GM, Weeks, JE, Oliver, JM, and Jones, MT. Athlete external load measures across a competitive season in high school basketball. J Strength Cond Res 37(11): 2206-2212, 2023-The purpose of this retrospective analysis was to quantify in-season external load and to determine if relationships existed between load metrics and basketball performance. Eleven male high school varsity basketball athletes (n = 11; mass 80.5 ± 9.6 kg, height 190.2 ± 9.4 cm, age 17.6 ± 0.7 years) were monitored across a season. PlayerLoad (PL), PL per minute (PL·min -1 ), total jumps, and explosive movements (EMs) were quantified using a commercially available local positioning unit. Basketball-specific performance metrics, including points scored, points allowed, point differentials, and shooting percentages for each quarter and game, were compiled. Data were analyzed using repeated-measure analysis of variance to evaluate differences in load by starting status, session type, game outcome, and game type. Pearson's correlation coefficients were used to assess relationships between load metrics and basketball performance. Statistical significance was set at p < 0.05. The mean values across 23 games for PL, PL·min -1 , total jumps, and EMs were 457 ± 104 AU, 10.9 ± 1.6 AU, 42.6 ± 9.6, and 46.7 ± 7.2, respectively. Relationships were observed ( p < 0.05) between PL and points scored ( r = 0.38) and free throw percentage ( r = 0.21). Further relationships were observed between PL·min -1 and free throw shooting percentage ( r = -0.27), and between points scored and total jumps ( r = 0.28), and EMs ( r = 0.26). Notable differences in game demands were observed for playing status. Meaningful differences in measures of external load were observed between each quarter of play, with the highest measures evident in quarters 1 and 3. Guards and forwards experienced minimal differences in external load during gameplay, and game outcome did not result in differences. Higher point totals corresponded with higher PL, total jumps, and EM.

First Report of Bacterial Wilt Disease Caused by Ralstonia solanacearum on Southern Highbush Blueberries (Vaccinium corymbosum interspecific hybrids) in Georgia U.S.A.

Members of the Ralstonia solanacearum (Rs) species complex have recently been reported to cause bacterial wilt on southern highbush (SHB) blueberries in Florida (Norman et al. 2018), a disease first reported on blueberry (northern highbush; Vaccinium corymbosum) in New Jersey (Patel et al. 2013). SHB blueberries are widely grown in the southern United States, and SHB cultivars represent the majority of the blueberry acreage in Georgia - the U.S. state with the largest blueberry acreage (NASS 2022). In Fall 2020, three-year old SHB plants (cv. 'Indigocrisp') showing leaf bronzing, wilting, and dieback were collected from two field sites in Clinch County, GA. At these locations, numerous plants were rapidly dying, with symptoms appearing to have spread down rows. Plant material tested positive using the ImmunoStrip® for Rs (Agdia, Inc., Elkhart, IN). From one location, the remaining sample was submitted to USDA-APHIS Select Agent Services who determined that Rs was present but a select agent (Rs Race 3, Biovar 2) was not. Following this, six adjacent, symptomatic SHB plants were collected from the same field location. These plants tested positive using the ImmunoStrip® for Rs, and red-pigmented mucoid colonies typical of Rs formed within 48 h at 28°C on triphenyltetrazolium chloroide (TZC) isolation medium (Kelman 1954). DNA was extracted from pure Rs cultures using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1987) and tested using polymerase chain reaction (PCR). Primers pairs AMB013/AMB014 (Fegan and Prior 2005) and ENDO-F/ENDO-R (Ji et al. 2007) were used to amplify 558 nt and 843 nt portions of the 16S rRNA region and Rs endoglucanase gene, respectively. Resulting amplicons were purified using an E.Z.N.A.® Cycle Pure Kit (Omega Bio-Tek, Norcross, GA), Sanger sequenced in both directions (Eurofins Genomics, Louisville, KY), and compared to publicly available Rs sequences in Genbank. The 16 rRNA sequence from all obtained isolates (accession ON938207) had 100% identity to Rs strain CFBP2957 (FP885897), while the endoglucase sequence (ON938206) had 100% identity to phylotype IIa, sequevar 5 Rs strain CIP-426 (MF461810) and phylotype IIa, sequevar 39 Rs strain 19-058 (MT314067), among others. To fulfill Koch's postulates, an isolate ('Ral21-1') was grown on TZC medium for 48 h at 28°C and suspended in 8.5 g/L NaCl at 1 x 108 CFU/ml. Five young, tissue cultured SHB plants (cv. 'Kestrel') in 25 cm pots were drenched with 50 ml of Rs suspension. For six weeks, plants were maintained in the greenhouse at 21-32°C. Typical bacterial wilt symptoms (leaf bronzing/scorching) developed in all inoculated plants, and infections were confirmed using Immunostrip®. Rs was reisolated and confirmed via PCR and sequencing as previously described. While Ralstonia has been known to cause disease on numerous crops in Georgia, this represents a first report of bacterial wilt in Georgia blueberries. Relative to rabbiteye blueberries (V. virgatum), recent reports suggest that SHB are much more susceptible to bacterial wilt (Conner et al. 2022). Accordingly, given the transition from rabbiteye to SHB within Georgia's blueberry production region over the past two decades and the ability of Rs to spread easily in water, soil, or via infected plant material, the presence of this disease within the state represents a significant threat to blueberry production. Additional characterization of Ralstonia isolates from Georgia may help assess the risk of future outbreaks.

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the XfmrpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Growth Dynamics and Antibiotic Elimination of Symbiotic Rickettsia buchneri in the Tick Ixodes scapularis (Acari: Ixodidae).

Rickettsia buchneri is the principal symbiotic bacterium of the medically significant tick Ixodes scapularis This species has been detected primarily in the ovaries of adult female ticks and is vertically transmitted, but its tissue tropism in other life stages and function with regard to tick physiology is unknown. In order to determine the function of R. buchneri, it may be necessary to produce ticks free from this symbiont. We quantified the growth dynamics of R. buchneri naturally occurring in I. scapularis ticks throughout their life cycle and compared it with bacterial growth in ticks in which symbiont numbers were experimentally reduced or eliminated. To eliminate the bacteria, we exposed ticks to antibiotics through injection and artificial membrane feeding. Both injection and membrane feeding of the antibiotic ciprofloxacin were effective at eliminating R. buchneri from most offspring of exposed females. Because of its effectiveness and ease of use, we have determined that injection of ciprofloxacin into engorged female ticks is an efficient means of clearing R. buchneri from the majority of progeny.IMPORTANCE This paper describes the growth of symbiotic Rickettsia buchneri within Ixodes scapularis through the life cycle of the tick and provides methods to eliminate R. buchneri from I. scapularis ticks.

Interspecies bacterial communication produces a delicate balance between Vibrio cholerae and the chironomid egg mass microbiome.

The evolution of antimicrobial resistance in bacterial pathogens is considered by the World Health Organization to be one of the ten most concerning public health threats facing humanity (World Health Organization, 2020). Bacterial diseases previously controllable by antibiotics are resurging and treatment options are dwindling. Cholera is one such disease. Human pathogenic strains of Vibrio cholerae cause as many as 4 million cases of disease resulting in over 100,000 deaths each year (Ali et al. 2015) and multidrug-resistant V. cholerae is now established where pandemic cholera persists. Vibrio cholerae is fundamentally an aquatic species thriving in brackish and estuarial waters. Its environmental prevalence, together with both extracellular and intracellular infection of alternative arthropod and mollusc hosts, produces a highly complex ecological milieu that is not well understood. With the absence of reliable antibiotic-based treatment options, it is necessary to build a better understanding of V. cholerae biology and ecology in order to develop alternative methods for risk modelling and disease control. In this issue of Molecular Ecology, authors Sela, Hammer, and Halpern experimentally investigated a mechanism by which V. cholerae pathogenicity is affected by interspecies quorum sensing involving an array of bacterial species from the microbiome of an alternative arthropod host, the egg mass of a chironomid midge (Diptera:Chironomidae) (Sela et al. 2020). Quorum sensing is a mechanism whereby bacteria communicate with each other using autoinducers and is known to be important, for example, in shaping virulence in a variety of pathogenic bacteria. The innovative methodologies they used, both in molecular and protein biology and reductive investigative microbiomics, are helping to develop the tools needed for understanding this understudied ecological system and fighting cholera in a post-antibiotic world.

Blood Biomarkers of Sports-Related Concussion in Pediatric Athletes.

OBJECTIVE: To examine changes in blood biomarkers, serum neurofilament light (Nf-L), and plasma tau, as well as the relationship between blood biomarkers and symptom reports, in athletes with a sports-related concussion. DESIGN: Prospective cohort study. SETTING: Private community-based concussion clinic. PARTICIPANTS: Athletes aged 13 to 18 years old with a diagnosed sports-related concussion presenting to a concussion clinic within 7 days of injury and noninjured athletes with no history of concussion aged 13 to 23 years old. ASSESSMENT AND MAIN OUTCOME MEASURES: Injured athletes provided a blood sample at the initial clinical evaluation and again at least 6 months after injury. Noninjured athletes provided a single blood sample. All participants completed symptom reports during each visit. Statistical comparisons of biomarker concentrations and symptom reports were conducted. RESULTS: The mean rank for tau was significantly lower for concussed athletes compared with nonconcussed athletes. In contrast, the mean rank of Nf-L was higher for concussed athletes than for nonconcussed athletes, although the difference was nonsignificant. Plasma tau was significantly lower postinjury compared with 6 months after injury, whereas serum Nf-L was significantly higher postinjury. There was a weak but significant inverse relationship observed between tau and the number of symptoms reported, but no relationship was observed between Nf-L and the number of symptoms reported. CONCLUSIONS: These data indicate that in the days following a sports-related concussion, the blood biomarkers tau and Nf-L display contrasting patterns of change but may not be related to self-reported symptom scores.

Correction to: Genome-enabled insights into the biology of thrips as crop pests.

An amendment to this paper has been published and can be accessed via the original article.

Genome-enabled insights into the biology of thrips as crop pests.

BACKGROUND: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. RESULTS: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. CONCLUSIONS: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

First Report of Citrus tristeza virus Infecting Citrus Trees in Georgia, USA.

Citrus tristeza virus (CTV) [genus Closterovirus; family Closteroviridae] is one of the most important, economically devastating viruses of citrus worldwide. On citrus trees grafted onto sour orange rootstock, typical CTV symptoms include dieback and defoliation, stunting, curling and chlorotic leaves, stem-pitting, and pinholes below the bud union on the inner face of the bark (Moreno et al. 2008). This single-stranded, positive-sense RNA virus is most efficiently transmitted by the brown citrus aphid (Toxoptera citricida), but it can also be transmitted by other aphid species and through grafting of infected plant material onto healthy plants (Moreno et al 2008; Herron et al. 2006). In Fall 2020, leaf material for virus testing was collected from 13 navel orange trees (Citrus × sinensis) grafted onto Poncirus trifoliata rootstocks (including 'Flying Dragon') located in a citrus research orchard in Tifton, GA. Trees ranged in age from 2 to 10 years, with the younger trees having been grafted from cuttings taken from the older trees. The oldest of these trees was derived from cuttings taken in 2009 from an orange tree growing locally in a residential yard in Tifton; this parent tree was more than 15 years old when these cuttings were obtained and was no longer available for sampling as of 2020. Symptoms or other visible signs of disease had not been noted on any of the tested trees, and trees were chosen for testing prior to the further dissemination of this plant material. The presence of CTV was verified via molecular and serological testing. CTV infection was initially confirmed in 8 of 13 tested samples using the ImmunoStrip® for CTV assay (Agdia® Inc., Elkhart, IN, cat no: ISK 78900/0025) according to the manufacturer's instructions. RNA was extracted from leaf material collected from the 13 sampled trees using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Following cDNA synthesis, samples were tested for the presence of CTV by reverse-transcription PCR using primer pair AR18F (5'-ATGTCAGGCAGCTTGGGAAATT-3') and AR18R (5'-TTCGTGTCTAAGTCRCGCTAAACA-3') which produces a 511 bp amplicon (Roy et al., 2005). PCR reactions confirmed the presence of CTV, with the same eight samples that had previously tested positive via Immunostrip® producing PCR fragments of the expected size. Amplified products from two of these samples were then sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and subjected to BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for further identification. Sequence analysis revealed that the obtained partial sequences (MW540805) from the p18 gene of both isolates were 100% identical to one another and shared 100% identity to corresponding sequences from CTV strain N4 (MK779711.1). To the best of our knowledge, this is the first report of CTV infecting citrus plants in Georgia. CTV could pose an imminent threat to the emerging citrus industry in Georgia if it were to become established in commercial citrus plantings either via the dissemination of infected plant material or via vector transfer of the virus under field conditions. While the brown citrus aphid is not known to be widespread in Georgia at this time, other CTV vectors are prevalent including the cotton aphid (Aphis gossypii) and the black citrus aphid (T. aurantia). Georgia citrus growers and plant propagators should be aware of this virus and take appropriate control measures to prevent the spread of this viral diseas.

Body Composition and Bone Mineral Density of Division 1 Collegiate Track and Field Athletes, a Consortium of College Athlete Research (C-CAR) Study.

The purpose of the present study was to generate normative values for total and regional body composition in male and female Division 1 collegiate track and field athletes using dual X-ray absorptiometry. We also sought to examine body composition by event and sex. Data were used from the Consortium of College Athlete Research group. A total of 590 (male [M]/female [F] = 274/316) athletes had their height, weight, total and regional fat mass (FM), lean mass, and bone mineral density (BMD) measured. Athletes were classified into 1 of 7 categories: Jumps (M/F = 28/30); Long Distance (M/F = 104/110), Middle Distance (M/F = 27/24), Multievent (M/F = 11/9), Pole Vault (M/F = 21/27), Sprints (M/F = 54/96), and Throws (M/F = 29/20). Total and regional differences between events and sex were assessed by analysis of variance. Except for male and female throwers, all other track and field athletes' mean percent body fat (M: 10.3%-12.6%, F: 17.5%-21.6%) and visceral FM (<500 g) was low, but in a healthy range. As expected, throwers had significantly (p < 0.05) higher total and regional FM and lean mass than other events. In addition, male (1.55 ± 0.11 g/cm2) and female (1.40 ± 0.12 g/cm2) throwers had significantly greater BMD than all other events while male (1.25 ± 0.10 g/cm2) and female (1.16 ± 0.09 g/cm2) distance runners had significantly lower BMD than all other events. In conclusion, track athletes' body composition differed across events for both males and females. These measurements provide normative data on NCAA Division 1 male and female track and field athletes.

Body Composition of National Collegiate Athletic Association (NCAA) Division I Female Soccer Athletes through Competitive Seasons.

The purpose of this study was to examine body composition of National Collegiate Athletic Association Division I female soccer players by position and season. One hundred seventy-five female athletes were categorized by positions of forward (n=47), midfielder (n=51), defender (n=57), and goalkeeper (n=20). A dual X-ray absorptiometry scan assessed percent body fat, total lean mass, total fat mass, arm and leg lean mass and fat mass, and visceral adipose tissue. Goalkeepers had significantly higher total, arm, and leg lean mass and fat mass compared to all other positions (p<0.05). For seasonal changes, body fat percentage was significantly higher in winter off-season (26.7%) compared to summer off-season (25.7%) and pre-season (25.8%; p<0.01) for all positions. Total and leg lean mass was significantly lower in winter off-season compared to all other seasons, and total lean mass was significantly higher in summer off-season than pre-season (p<0.01). Overall, goalkeepers were significantly different than all other positions. Body fat percentage increased and lean mass decreased in winter off-season indicating potential undesired changes in training and/or nutrition over the break whereas lean mass was the highest in summer off-season potentially reflecting the emphasis on resistance training and increased volume of training.

Total and Regional Body Composition of NCAA Division I Collegiate Basketball Athletes.

This study aimed to examine body composition using dual X-ray absorptiometry (DXA) in male and female NCAA Division I collegiate basketball athletes. Two-hundred ten (male [M]/female [F]=88/122) basketball athletes' total and regional fat mass, lean mass, bone mineral density, and visceral adipose tissue were measured. Athletes were classified as: point guards (M/F=27/34), shooting guards (M/F=18/27), small forwards (M/F=13/18), power forwards (M/F=21/27), and centers (M/F=9/16). ANOVA and Tukey's HSD assessed positional differences by sex. In males, centers and power forwards had greater total fat mass (p<0.025), lean mass (p≤0.001), and visceral adipose tissue (p<0.001) than other positions. Male centers had greater arm and leg fat mass and lean mass than point guards, shooting guards, and small forwards (p≤0.049), and greater arm bone mineral density than point guards (p=0.015). In females, centers had greater total fat mass (p<0.001) vs. other positions and greater total lean mass, arm fat and lean masses, arm and leg bone mineral density, and visceral adipose tissue vs. point guards and shooting guards (p≤0.005). Female point guards had lower total bone mineral density than power forwards (p=0.008). In conclusion, these sex- and position-specific total and regional body composition measurements in collegiate basketball players provide population-specific normative data.

Natural Infection of Southern Highbush Blueberry (Vaccinium corymbosum Interspecific Hybrids) by Xylella fastidiosa subsp. fastidiosa.

Xylella fastidiosa (Xf) is an emerging insect-vectored, xylem-limited bacterium that can cause disease on several economically important fruit and tree crops including almond, blueberry, citrus, grapevine, peach, and pecan. On blueberry, Xf causes bacterial leaf scorch (BLS), which is prevalent in the southeastern United States. This disease, previously reported to be caused by Xf subsp. multiplex (Xfm), can result in rapid plant decline and death of southern highbush (SHB) blueberry cultivars. In 2017, a survey of blueberry plantings in southern Georgia (U.S.A.) confirmed the presence of Xf-infected plants in eight of nine sites examined, and seven isolates were cultured from infected plants. Genetic characterization of these isolates through single-locus and multilocus sequence analysis revealed that three isolates from two sites belonged to Xf subsp. fastidiosa (Xff), with significant similarity to isolates from grapevine. After these three isolates were artificially inoculated onto greenhouse-grown SHB blueberries (cv. 'Rebel'), symptoms typical of BLS developed, and Xff infection was confirmed through genetic characterization and reisolation of the bacterium to fulfill Koch's postulates. Because all previously reported Xf isolates from blueberry have been characterized as Xfm, this is the first time that isolation of Xff has been reported from naturally infected blueberry plantings. The potential impact of Xff isolates on disease management in blueberry requires further exploration. Furthermore, given that isolates from both Xfm and Xff were obtained within a single naturally infected blueberry planting, blueberry in southern Georgia may provide opportunities for intersubspecific recombination between Xff and Xfm isolates.

First Report of Hop stunt viroid Infecting Citrus Trees in Georgia, USA.

In recent years, citrus production has rapidly increased within the state of Georgia (USA), and there are now citrus plantings within at least 32 counties in residential, production, and nursery settings. Among the pathogens capable of infecting citrus are viroids, the smallest plant pathogens. Viroids are comprised of circular, single-stranded RNA ranging from 246-463 nucleotides in length (Ito et al., 2002). Hop stunt viroid (HSVd) is one of several viroids known to infect citrus. This viroid has been previously reported within Arizona, California, Florida, Texas, and Washington in the United States and in other locations throughout the world (Hadidi, 2017). HSVd is often spread mechanically on contaminated tools or through grafting. With a wide host range that includes the families Moraceae, Rosaceae, and Rutaceae (citrus), this viroid can easily move throughout a nursery and spread to other plants (Hadidi, 2017). Symptoms of HSVd include a discoloration and gumming of phloem tissues, stem pitting, bark splitting, and chlorotic and stunted growth in susceptible citrus varieties including tangerines and their hybrids (Hadidi, 2017). There are not typically symptoms on leaves or fruits; however, lime plants have shown some yellowing on leaves (Hadidi, 2017). In May and June of 2020, leaf samples were collected from 12 different citrus plants in nursery settings in Berrien and Mitchell counties in Georgia. The cultivars sampled from Citrus reticulata 'Dekopon'. The sampled trees looked relatively healthy with little or no signs of damage, but were selected for testing to ensure that they were viroid free. Reverse transcription-polymerase chain reaction (RT-PCR) was initially used to verify infection with HSVd. Genomic RNA was extracted from the leaf tissue of twelve plants using the TRIzol reagent (Thermofisher, Waltham, MA). Following cDNA synthesis, samples were tested for the presence of HSVd using the primer pair HSVd-F (5'-GGCAACTCTTCTCAGAATCCAGC-3') and HSVd-R (5'-CCGGGGCTCCTTTCTCAGGTAAGT-3') which produces a 302 bp amplicon (Sano et al., 1988). The PCR reactions for nine of the tested samples did not result in the production of any bands, however the other three samples, all Citrus reticulata 'Dekopon', produced the expected amplicon for HSVd. The amplified products were sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and the identity of the fragment sequences was confirmed using BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Partial sequences from these amplicons (deposited as accession number MT632007) shared 99% identity to corresponding HSVd sequences in Genbank (accession number MG779542). In addition to RT-PCR and sequencing, the recombinase-polymerase-amplification (RPA) technology based AmplifyRP® Acceler8™ end-point detection assay (Agdia® Inc., Elkhart, IN) was performed on previously confirmed tissue according to the manufacturer's instructions. This assay also confirmed the presence of HSVd viroid in the three samples that had been previously confirmed via RT-PCR. To the best of our knowledge, this is the first report of HSVd infecting Citrus reticulata 'Dekopon' in Georgia. If this viroid were to spread within the growing Georgia citrus industry, it could pose a significant threat to citrus plantings that contain susceptible varieties. Nursery stock infected with this viroid should be destroyed, and Georgia nursery producers and citrus growers should take appropriate precautions to prevent the spread of this viroid disease, including properly sanitizing tools used for citrus grafting and pruning. Further research is needed to determine the distribution of HSVd and its potential to impact commercial citrus production in Georgia.

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus.

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.

Rest Redistribution Does Not Alter Hormone Responses in Resistance-Trained Women.

Merrigan, JJ, Tufano, JJ, Fields, JB, Oliver, JM, and Jones, MT. Rest redistribution does not alter hormone responses in resistance-trained women. J Strength Cond Res 34(7): 1867-1874, 2020-The purpose was to examine acute effects of rest redistribution (RR) on perceptual, metabolic, and hormonal responses during back squats. Twelve resistance-trained women (training age 5 ± 2 years; one repetition maximum [1-RM] per body mass, 1.6 ± 0.2) performed traditional (TS, 4 sets of 10 repetitions with 120 seconds interset rest) and RR sets (4 sets of two 5 repetition clusters with 30-second intraset rest and 90-second interset rest) in counterbalanced order, separated by 72 hours. Both conditions were performed at 70% 1RM with 360 seconds of total rest. Ratings of perceived exertion (RPE) were taken after each set. Blood was sampled at baseline, after each set, and at 5, 15, 30, and 60 minutes, as well as 24 and 48 hours after training. Alpha level was p ≤ 0.05. The RPE progressively increased throughout both conditions (p = 0.002) with a greater overall mean for TS (5.81 ± 0.14) than RR (4.71 ± 0.14; p = 0.003). Lactate increased above baseline and remained elevated through 15 minutes post in both conditions (4.00 ± 0.76; p = 0.001), with greater lactate levels for TS (6.33 ± 0.47) than RR (4.71 ± 0.53; p < 0.001). Total testosterone was elevated after set 2 (0.125 ± 0.02; p = 0.011), but no other time point, while free testosterone remained unchanged. Growth hormone continually rose from baseline to set 3 and returned to baseline by 60 minutes post (20.58 ± 3.19). Cortisol and creatine kinase did not change over time. No condition × time interactions existed for any hormone (p > 0.05). Use of rest redistribution resulted in lower perceived effort and lactate responses. Yet, hormone responses during rest redistribution were no different from TS.

Positional Body Composition of Female Division I Collegiate Volleyball Players.

Bisch, KL, Bosch, TA, Carbuhn, A, Stanforth, PR, Oliver, JM, Bach, CW, and Dengel, DR. Positional body composition of female division I collegiate volleyball players. J Strength Cond Res 34(11): 3055-3061, 2020-The primary study objective was to measure positional differences in total and regional body composition among female NCAA Division I collegiate volleyball players using dual X-ray absorptiometry (DXA). The secondary objective was to examine normative age curves for fat and lean mass (LM) variables. Ninety female volleyball players from 5 universities received a DXA scan. Athletes were categorized by position: middle blocker (MB = 31), outside hitter (OH = 32), setter (ST = 9), and Libero (LB = 18). Height, body mass, total and regional fat mass (FM), LM, bone mineral density (BMD), and abdominal visceral adipose tissue were measured by DXA. Body mass distribution ratios were calculated. The secondary age analysis included a subset of 153 DXA scans (n = 83, ages 18-21 years). Front row players (i.e., MB and OH) had significantly greater total and regional LM and BMD measures (p < 0.05, all), compared with non-front row players (i.e., LB and ST). Differences in total LM (p < 0.001) were significantly influenced by height. Front row players had consistently lower mass distribution ratios compared with non-front row players (p < 0.05, all). Lean mass index (LMI, p = 0.752) and FM index (FMI, p = 0.392) were not significantly different across ages. Back row players have greater relative upper body mass, whereas mass in front row players is more evenly distributed between the upper and lower body. Bone mineral density differences may be influenced by repeated impact of jumping during the attacking and blocking actions of front row players. Minimal changes in LMI and fluctuations in FMI can be expected across an athlete's career.

Ehrlichia Isolate from a Minnesota Tick: Characterization and Genetic Transformation.

Ehrlichia muris subsp. eauclairensis is recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species of Ehrlichia The isolate could be grown in a variety of cultured cell lines and was effectively transmitted between Ixodes scapularis ticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues. Conversely, Amblyomma americanum ticks were not susceptible to infection by the Ehrlichia Histologic sections further revealed that the wild-type isolate was highly virulent for mice and hamsters, causing severe systemic disease that was frequently lethal. A Himar1 transposase system was used to create mCherry- and mKate-expressing EmCRT mutants, which retained the ability to infect rodents and ticks.IMPORTANCE Ehrlichioses are zoonotic diseases caused by intracellular bacteria that are transmitted by ixodid ticks. Here we report the culture isolation of bacteria which are closely related to, or the same as the Ehrlichia muris subsp. eauclairensis, a recently recognized human pathogen. EmCRT, obtained from a tick removed from deer at Camp Ripley, MN, is the second isolate of this subspecies described and is distinctive in that it was cultured directly from a field-collected tick. The isolate's cellular tropism, pathogenic changes caused in rodent tissues, and tick transmission to and from rodents are detailed in this study. We also describe the genetic mutants created from the EmCRT isolate, which are valuable tools for the further study of this intracellular pathogen.