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Phyllosticta citricarpa is the causative agent of citrus black spot (CBS), a cosmetic fungal disease that has been reported in most of the citrus-growing regions of the world. The occurrence of CBS in orchards is predominantly controlled by the application of fungicides during the fruit susceptibility period. Benzimidazoles (benomyl and carbendazim) and toluamides (ethaboxam and zoxamide) are two systemic fungicide classes suitable for the control of CBS in South Africa. Both inhibit proper assembly of the fungal β-tubulin protein during mitosis, and resistance to these fungicides is primarily related to alterations in the binding sites on this target protein. In this study, P. citricarpa isolates with known resistance status were subjected to whole genome sequencing, the mutations conferring resistance to benomyl and zoxamide were examined and allele-specific primers targeting these mutations were developed. The allele-specific multiplex PCR assay for the detection of benomyl resistance in P. citricarpa will eliminate the need for laborious and time-consuming sensitivity assays or DNA sequencing.

Funding: This paper presents independent research supported by the National Funding: Institute for Health Research (NIHR) Clinical Research Facility and the NIHR Biomedical Research Centre based at Imperial College Healthcare NHS Trust. AA is supported by an NIHR Clinician Scientist award (CS‐2018‐18‐ST2‐002). MAM is funded by the Tommy’s National Centre for Miscarriage Research. MP is supported by an NIHR Academic Clinical Lectureship. CI-E is supported by an Imperial College-BRC IPPRF Fellowship. SAC is supported by an NIHR Academic Clinical Lectureship. EGM is supported by an MRC clinical training fellowship (MR/T006242/1). LY is supported by an MRC clinical training fellowship (MR/R000484/1). ANC is supported by the NHS and BRC. TB is supported by the NIHR Biomedical Research Centre based at Imperial College Healthcare NHS Trust. WSD is supported by an NIHR Research Professorship (RP-2014-05-001). Context: Antenatal complications such as hypertensive disorders of pregnancy (HDP), fetal growth restriction (FGR), gestational diabetes (GDM), and preterm birth (PTB) are associated with placental dysfunction. Kisspeptin has emerged as a putative marker of placental function, but limited data exist describing circulating kisspeptin levels across all three trimesters in women with antenatal complications. Objective: To assess whether kisspeptin levels are altered in women with antenatal complications. Methods: Women with antenatal complications (n = 105) and those with uncomplicated pregnancies (n = 265) underwent serial ultrasound scans and blood sampling at the Early Pregnancy Assessment Unit at Hammersmith Hospital, UK, at least once during each trimester (March 2014 to March 2017). The women with antenatal complications (HDP [n = 32], FGR [n = 17], GDM [n = 35], PTB [n = 11], and multiple complications [n=10]) provided 373 blood samples and the controls provided 930 samples. Differences in circulating kisspeptin levels were assessed. Participants: Women with antenatal complications: HDP (n=32), FGR (n=17), GDM (n=35) and PTB (n=11), and 10 women with multiple complications, provided 373 blood samples, and a further 265 controls provided 930 samples. Results: Third-trimester kisspeptin levels were higher than controls in HDP but lower in FGR. The odds of HDP adjusted for gestational age, maternal age, ethnicity, BMI, smoking, and parity were increased by 30% (95% CI, 16%-47%; P

Citrus black spot (CBS), caused by Phyllosticta citricarpa, is a disease that affects citrus worldwide. In different regions of the world where both mating types occur, reports differ as to whether asexually produced pycnidiospores play an important role in the epidemiology of CBS and fruit infections. Therefore, we investigated the potential role of pycnidiospores in two lemon orchards in South Africa by using microsatellite-based analysis of fruit populations over time (two seasons) and space (distance). The two orchards were situated in the semiarid North West province (NW) and subtropical Mpumalanga province (MP). Each population contained both mating types in 1:1 ratios, and linkage disequilibrium analysis indicated a random mating population. A total of 109 and 94 multilocus genotypes (MLGs) were detected across the two seasons in the NW and MP orchards, respectively. Psex analyses indicated that most MLGs probably resulted from sexual reproduction, but there were six predominant MLGs in each orchard that were probably replicated via asexual reproduction. Each of the predominant MLGs was monomorphic for mating type. In the NW, five predominant and widespread MLGs caused 46 and 44% of the fruit infections in the two seasons, whereas in MP, three MLGs caused 34 and 48% of the infections. Asexual reproduction in both orchards was supported by low MLG evenness values in all populations. In both orchards, distance was not a reliable predictor of population genetic substructuring or season. Populations of P. citricarpa in the MP and NW orchards were significantly genetically differentiated from each other.

Phyllosticta citricarpa is a fungal pathogen causing citrus black spot (CBS). As a regulated pest in some countries, the presence of the pathogen limits the export of fruit and is therefore of agricultural and economic importance. In this study, we used high throughput sequencing data to infer the global phylogeographic distribution of this pathogen, including 71 isolates from eight countries, Argentina, Australia, Brazil, China, Cuba, Eswatini, South Africa and the United States of America. We assembled draft genomes and used a pairwise read mapping approach for the detection and enumeration of variants between isolates. We performed SSR marker discovery based on the assembled genome with the best assembly statistics, and generated genotype profiles for all isolates with 1987 SSR markers in silico. Furthermore, we identified 32,560 SNPs relative to a reference sequence followed by population genetic analyses based on the three datasets; pairwise variant counts, SSR genotypes and SNP genotypes. All three analysis approaches gave similar overall results. Possible pathways of dissemination among the populations from China, Australia, southern Africa and the Americas are postulated. The Chinese population is the most diverse, and is genetically the furthest removed from all other populations, and is therefore considered the closest to the origin of the pathogen. Isolates from Australia, Eswatini and the South African province Mpumalanga are closely associated and clustered together with those from Argentina and Brazil. The Eastern Cape, North West, and KwaZulu-Natal populations in South Africa grouped in another cluster, while isolates from Limpopo are distributed between the two aforementioned clusters. Southern African populations showed a close relationship to populations in North America, and could be a possible source of P. citricarpa populations that are now found in North America. This study represents the largest whole genome sequencing survey of P. citricarpa to date and provides a more comprehensive assessment of the population genetic diversity and connectivity of P. citricarpa from different geographic origins. This information could further assist in a better understanding of the epidemiology of the CBS pathogen, its long-distance dispersal and dissemination pathways, and can be used to refine phytosanitary regulations and management programmes for the disease.

Citrus tatter leaf virus (CTLV), a strain of apple stem grooving virus, is a virus of citrus that is of commercial importance for trifoliate orange (Poncirus trifoliata) and trifoliate hybrid rootstocks. Bud-union crease is a CTLV-induced incompatibly symptom observed with these rootstocks and citrus scions which results in stunted tree growth. CTLV is a regulated pathogen for most citrus budwood certification programs worldwide. Due to the requisite virus testing, commercially supplied scion budwood would therefore seldom be a point of contamination. However, citrus rootstocks are primarily propagated by seed and the potential for seed transmission of CTLV in citrus is unclear and remains a potential source of infection. A single, unconfirmed report of seed transmission of CTLV in ‘Eureka’ lemon (Citrus × limon) exists, and although this report was not of a rootstock cultivar, it does point to the risk of seed transmission in citrus and close relatives in the family Rutaceae. CTLV-positive ‘Meyer’ lemon (C. × limon) trees in an experimental plot in the Eastern Cape Province of South Africa presented an opportunity to again test seed transmission in citrus. Seed was obtained from the infected ‘Meyer’ lemon trees, and 1164 seedlings were grown in an insect-secure greenhouse and screened for CTLV in pooled batches by conventional and real-time RT-PCR. No positive samples were detected and results indicated a lack of virus seed transmission in ‘Meyer’ lemon. Findings of this study are therefore not supportive of CTLV seed transmission in citrus.

OBJECTIVES: Our primary aim was to report the incidence of enhanced myometrial vascularity (EMV) in consecutive women attending our early pregnancy assessment unit, following first-trimester miscarriage. We aimed further to evaluate the clinical presentation and complications associated with expectant and surgical management of EMV in these women. METHODS: This was a prospective cohort study conducted in a London teaching hospital between June 2015 and June 2018, including consecutive patients with an observation of EMV on transvaginal ultrasonography following first-trimester miscarriage. The diagnosis was made following the subjective identification of EMV using color Doppler ultrasonography and a peak systolic velocity (PSV) ≥ 20 cm/s within the collection of vessels. Women were followed up with repeat scans every 14 days. Management was expectant unless intervention was indicated because of excessive or prolonged bleeding, persistent presence of retained tissue in the endometrial cavity or patient choice. The final clinical outcome was recorded. Time to resolution of EMV was defined as the interval from detection of EMV until resolution. RESULTS: During the study period, there were 2627 first-trimester fetal losses in the department and, of these, 40 patients were diagnosed with EMV, hence the incidence of EMV following miscarriage was 1.52%. All cases were associated with ultrasound evidence of retained products of conception (RPOC) at presentation (mean dimensions, 22 × 20 × 20 mm). Thirty-one patients opted initially for expectant management, of which 18 had successful resolution without intervention, five were lost to follow-up and eight subsequently had surgical evacuation due to patient choice. No expectantly managed case required emergency intervention. Nine patients chose surgical evacuation as primary treatment. No significant correlation was seen between PSV within the EMV at presentation and blood loss at surgery. Median PSV was 47 (range, 20-148) cm/s. The estimated blood loss in all cases managed surgically ranged from 20-300 mL. Presence of RPOC was confirmed in all specimens that were sent for analysis following surgery. For cases successfully managed expectantly, the mean time to resolution was 48 (range, 21-84) days. In the nine cases managed surgically from the beginning, the mean time to resolution of EMV was 10.6 (range, 3-29) days. CONCLUSIONS: This study suggests that EMV is an uncommon finding following miscarriage and is associated with the presence of RPOC. Expectant management was a safe option in our cohort, with minimal bleeding, although it was associated with protracted time to resolution. In patients who opted for surgery, the maximum blood loss was 300 mL and no patient required blood transfusion or embolization. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd. ispartof: ULTRASOUND IN OBSTETRICS & GYNECOLOGY vol:55 issue:5 pages:676-682 ispartof: location:England status: published

OBJECTIVE: To assess whether sonographic diagnosis of intrauterine hematoma (IUH) in the first trimester of pregnancy is associated with first-trimester miscarriage and antenatal, delivery and neonatal complications. METHODS: This was a prospective observational cohort study of women with an intrauterine singleton pregnancy between 5 and 14 weeks' gestation recruited at Queen Charlotte's and Chelsea Hospital, London, UK, between March 2014 and March 2016. Participants underwent serial ultrasound examinations in the first trimester, and the presence, location, size and persistence of any IUH was evaluated. First-trimester miscarriage was defined as pregnancy loss before 14 weeks' gestation. Clinical symptoms, including pelvic pain and vaginal bleeding, were recorded at each visit using validated symptom scores. Antenatal, delivery and neonatal outcomes were obtained from hospital records. Logistic regression analysis and the chi-square test were used to assess the association between the presence and features of IUH and the incidence of adverse pregnancy outcome. Odds ratios (OR) were first adjusted for maternal age (aOR) and then further adjusted for the presence of vaginal bleeding or pelvic pain in the first trimester. RESULTS: Of 1003 women recruited to the study, 946 were included in the final analysis and of these, 268 (28.3%) were diagnosed with an IUH in the first trimester. The presence of IUH was associated with the incidence of preterm birth (aOR, 1.94 (95% CI, 1.07-3.52)), but no other individual or overall antenatal, delivery or neonatal complications. No association was found between the presence of IUH in the first trimester and first-trimester miscarriage (aOR, 0.81 (95% CI, 0.44-1.50)). These findings were independent of the absolute size of the hematoma and the presence of vaginal bleeding or pelvic pain in the first trimester. When IUH was present in the first trimester, there was no association between its size, content or position in relation to the gestational sac and overall antenatal, delivery and neonatal complications. Diagnosis of a retroplacental IUH was associated with an increased risk of overall antenatal complications (P = 0.04). CONCLUSIONS: Our findings demonstrate that there is no association between the presence of IUH in the first trimester and first-trimester miscarriage. However, an association with preterm birth, independently of the presence of symptoms of pelvic pain and/or vaginal bleeding, is evident. Women diagnosed with IUH in the first trimester should be counseled about their increased risk of preterm birth and possibly be offered increased surveillance during the course of their pregnancy. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd. ispartof: ULTRASOUND IN OBSTETRICS & GYNECOLOGY vol:55 issue:4 pages:536-545 ispartof: location:England status: published

A new species of dorylaimid nematode, Aporcelinus abeokutaensis sp. n., collected from a watermelon field in Nigeria, is described, illustrated and molecularly (D2-D3 28S ribosomal DNA) studied. It is characterized by its 1.18-1.52-mm-long body, lip region offset by weak constriction, 15.5-17.5 μm broad with perioral liplets, odontostyle 18-21.5 μm long at its dorsal side or 1.1-1.3 times the lip region diameter, neck 333-401 μm long, pharyngeal expansion occupying 45-51% of total neck length, uterus simple and 0.7-1.5 times the corresponding body diameter long, V = 48-53, tail conical with finely rounded tip (33-52 μm long, c = 26-41, c' = 1.3-1.9) and a variably distinct dorsal concavity, and male unknown. Molecular analysis reveals that the new species grouped with other species of Aporcelinus in a highly supported clade, confirming the monophyly of the genus.

Highlights • Interpretable machine learning models efficiently predict miscarriages. • Gradient boosted trees algorithm offers serious assets for clinical modeling. • Clinical predictive models’ interpretation can be improved with decision paths. • Post-hoc interpretably provides straightforward explanations for physicians., Background Clinical models to predict first trimester viability are traditionally based on multivariable logistic regression (LR) which is not directly interpretable for non-statistical experts like physicians. Furthermore, LR requires complete datasets and pre-established variables specifications. In this study, we leveraged the internal non-linearity, feature selection and missing values handling mechanisms of machine learning algorithms, along with a post-hoc interpretability strategy, as potential advantages over LR for clinical modeling. Methods The dataset included 1154 patients with 2377 individual scans and was obtained from a prospective observational cohort study conducted at a hospital in London, UK, from March 2014 to May 2019. The data were split into a training (70%) and a test set (30%). Parsimonious and complete multivariable models were developed from two algorithms to predict first trimester viability at 11–14 weeks gestational age (GA): LR and light gradient boosted machine (LGBM). Missing values were handled by multiple imputation where appropriate. The SHapley Additive exPlanations (SHAP) framework was applied to derive individual explanations of the models. Results The parsimonious LGBM model had similar discriminative and calibration performance as the parsimonious LR (AUC 0.885 vs 0.860; calibration slope: 1.19 vs 1.18). The complete models did not outperform the parsimonious models. LGBM was robust to the presence of missing values and did not require multiple imputation unlike LR. Decision path plots and feature importance analysis revealed different algorithm behaviors despite similar predictive performance. The main driving variable from the LR model was the pre-specified interaction between fetal heart presence and mean sac diameter. The crown-rump length variable and a proxy variable reflecting the difference in GA between expected and observed GA were the two most important variables of LGBM. Finally, while variable interactions must be specified upfront with LR, several interactions were ranked by the SHAP framework among the most important features learned automatically by the LGBM algorithm. Conclusions Gradient boosted algorithms performed similarly to carefully crafted LR models in terms of discrimination and calibration for first trimester viability prediction. By handling multi-collinearity, missing values, feature selection and variable interactions internally, the gradient boosted trees algorithm, combined with SHAP, offers a serious alternative to traditional LR models.

Citrus black spot (CBS), caused by

Study question Is there an association between serum oestradiol, vaginal microbial composition and pregnancy outcome in the early first trimester? Summary answer In women with a vaginal microbiome deplete of Lactobacillus species at the time of Pregnancy of Uncertain Viability (IPUV), higher serum oestradiol associates with livebirth. What is known already During pregnancy, oestradiol mediates vaginal mucosal properties and increases glycogen deposition in epithelial cells which is thought to support colonisation of Lactobacillus species. Low levels of Lactobacillus associates with adverse outcomes such as miscarriage and preterm birth. The direct relationship between systemic oestradiol and the vaginal microbiome has never been studied in pregnancy. However studies have shown a positive correlation between serum oestrone, vaginal glycogen and Lactobacillus abundance in menopausal women. Study design, size, duration This was a prospective cohort study where one-hundred women were recruited in early pregnancy at the time of IPUV and donated paired blood and vaginal samples. 40 women had an eventual miscarriage, 58 had a livebirth and two pregnancies were terminated. All 100 women donated one paired serum and vaginal sample at this time point, and 22 women with Lactobacillus depletion at the time of IPUV donated further longitudinal vaginal samples. Participants/materials, setting, methods Participants were recruited from an Early Pregnancy Unit and underwent transvaginal ultrasound assessment of their pregnancy. Serum samples were analysed with an immunoassay on a ROCHE COBAS E411 analyser for Oestradiol (pg/ml) and Progesterone (ng/ml). Bacterial DNA was extracted from paired vaginal swabs and sequenced using Illumina MiSeq sequencing of 16S rRNA gene amplicons. Main results and the role of chance Lactobacillus dominance of the vagina was associated with higher serum levels of E2 and progesterone compared to depletion (E2=398pg/ml vs 302pg/ml(p = 0.02), P4=23.1ng/ml vs 17ng/ml(p = 0.02)). E2 and P4 were positively correlated (r = 0.6, p Limitations, reasons for caution In this study, serum oestradiol levels were compared to the local vaginal bacterial environment. The ideal would be to study local vaginal oestradiol, glycogen and the bacterial composition. Wider implications of the findings: In contrast to previous studies in menopause where low oestrogen levels associate with the vaginal microbial composition, this study uses the high oestradiol environment of early pregnancy to study the mechanistic relationship between oestradiol and vaginal Lactobacillus abundance. Trial registration number NA

Supported by the National Institute for Health Research (NIHR) Clinical Research Facility and the NIHR Biomedical Research Centre based at Imperial College Healthcare National Health Services (NHS) Trust. The Section of Endocrinology and Investigative Medicine is funded by grants from the Medical Research Council and NIHR. A.A. is supported by an NIHR Clinician Scientist award (CS-2018-18-ST2-002). M.A.M. is supported by Tommy’s National Centre for Miscarriage Research . C.I.-E. is supported by an Imperial College-Biomedical Research Centre Imperial Post-doctoral, Post-CCT Research Fellowship. L.Y. is supported by an Medical Research Council Clinical Training Fellowship (MR/R000484/1). A.N.C. is supported by the NHS and Biomedical Research Centre. T.B. is supported by the NIHR Biomedical Research Centre based at Imperial College Healthcare NHS Trust. W.S.D. is supported by an NIHR Research Professorship (RP-2014-05-001). Objective: To compare the performance of kisspeptin and beta human chorionic gonadotropin (βhCG), both alone and in combination, as biomarkers for miscarriage throughout the first trimester. Design: Prospective, nested case-control study. Setting: Tertiary Centre, Queen Charlotte Hospital, London, United Kingdom. Patient(s): Adult women who had miscarriages (n = 95, 173 samples) and women with healthy pregnancies (n = 265, 557 samples).Intervention(s)The participants underwent serial ultrasound scans and blood sampling for measurement of plasma kisspeptin and βhCG levels during the first trimester. Main Outcome Measure(s): The ability of plasma kisspeptin and βhCG levels to distinguish pregnancies complicated by miscarriage from healthy pregnancies unaffected by miscarriage. Result(s): Gestation-adjusted levels of circulating kisspeptin and βhCG were lower in samples from women with miscarriages than in women with healthy pregnancies by 79% and 70%, respectively. The area under the receiver-operating characteristic curve for identifying miscarriage during the first trimester was 0.874 (95% confidence interval [CI] 0.844–0.904) for kisspeptin, 0.859 (95% CI 0.820–0.899) for βhCG, and 0.916 (95% CI 0.886–0.946) for the sum of the two markers. The performance of kisspeptin in identifying miscarriage improved with increasing length of gestation, whereas that of βhCG worsened. A decision matrix incorporating kisspeptin, βhCG, and gestational age had 83% to 87% accuracy for the prediction of miscarriage. Conclusion(s): Plasma kisspeptin is a promising biomarker for miscarriage and provides additional value to βhCG alone, especially during later gestational weeks of the first trimester. Publisher PDF

A new species of the genus Aporcella collected from a watermelon field in Nigeria is described, including its morphological and molecular (small subunit (SSU) and large subunit (LSU) ribosomal DNA (rDNA)) characterization. Aporcella femina sp. n. is distinguished by its 3.21-3.64 mm-long body, inner cuticle layer with fine but distinct transverse striation, lip region offset by deep constriction, 22-25 μm broad, odontostyle 20-26 μm, neck 661-811 μm long, pharyngeal expansion occupying 52-56% of the total neck length, female genital system didelphic-amphidelphic, uterus 191-350 μm or 1.9-3.3 mid-body diameters long, V = 52-57, tail short and convex conoid (35-48 μm, c = 72-98, c' = 0.7-0.9) and males absent. Phylogenetic analyses based on the partial sequence of SSU and LSU (D2-D3) rDNA revealed a close relationship of A. femina sp. n. with other Aporcella species, confirming the monophyly of the genus as well as its association to a clade made of several taxa characterized by the absence of pars refringens vaginae.

Citrus Black Spot (CBS), caused by the ascomycete, Phyllosticta citricarpa, is a fruit, foliar, and twig spotting fungal disease affecting the majority of commercial cultivars of citrus. The disease causes cosmetic lesions, may cause fruit drop and P. citricarpa is considered a quarantine pathogen by some countries, impacting domestic and international trade of citrus fruit. Regulatory requirements affecting fruit trade exist even though there is no documented case of disease spread via infected fruit into previously disease-free areas. To clarify the risk of fruit as a potential pathway for the spread of CBS, we developed a quantitative, probabilistic risk assessment model. The model provides an assessment of all steps in the fruit pathway, including production, packinghouse handling, transportation, export-import distribution channels, and consumer endpoints. The model is stochastic and uses Monte Carlo simulation to assess the risk of P. citricarpa moving through all steps in the pathway. We attempted to use all available literature and information to quantitate risk at each point in the potential pathway and by sequentially linking all steps to determine the overall quantitative risk. In addition, we assessed climatological effects on incidence of diseased fruit at production sites and on fungal reproduction and infection, as well as criteria for establishment at endpoints. We examined ten case studies between exporting and importing locations/countries. Model results indicated fruit to be an epidemiologically insignificant means for CBS spread, even between producing countries where CBS occurs and CBS-free importing countries with disease-conducive climates. We created a second model to examine the introduction of infected plant material from countries where CBS occurs. This model demonstrated significant probability of introduction via such infected material. However, pathogen establishment and disease development was still restricted only to areas with conducive climatological conditions. We created a tool to quantitatively explore the viability of various potential pathways via combinations of CBS-present production sites and corresponding pathway endpoints, including environments conducive and non-conducive to CBS. The tool is provided to aid decision makers on phytosanitary risk relative to international trade of citrus fruit.

Citrus black spot (CBS) is caused by Phyllosticta citricarpa, which is classified as a quarantine organism in certain countries whose concerns are that CBS-infected fruit may be a pathway for introduction of the pathogen. This study evaluated the reproductive capability and viability of P. citricarpa under simulated conditions in which the whole fruit, peel segments, or citrus pulp with CBS lesions were discarded. Naturally infected &lsquo, Midknight&rsquo, Valencia orange and &lsquo, Eureka&rsquo, lemon fruit, either treated using standard postharvest sanitation, fungicide, and wax coating treatments or untreated, were placed into cold storage for 5 weeks (oranges at 4 &deg, C and lemons at 7 &deg, C). Thereafter, treated and untreated fruit were incubated for a further 2 weeks at conditions conducive for CBS symptom expression and formation of pycnidia. The ability of pycnidia to secrete viable pycnidiospores after whole fruit and peel segments or peel pieces from citrus pulp were exposed to sunlight at warm temperatures (&plusmn, 28 &deg, C) and &plusmn, 75% relative humidity levels was then investigated. The combination of postharvest treatments and cold storage effectively controlled CBS latent infections (&gt, 83.6% control) and pycnidium formation (&lt, 1.4% of lesions formed pycnidia), and the wax coating completely inhibited pycnidiospore release in fruit and peel segments. Pycnidiospores were secreted only from lesions on untreated fruit and peel segments and at low levels (4.3&ndash, 8.6%) from peel pieces from pulped treated fruit. However, spore release rapidly declined when exposed to sunlight conditions (1.4% and 0% after 2 and 3 days, respectively). The generally poor reproductive ability and viability of CBS fruit lesions on harvested fruit, particularly when exposed to sunlight conditions, supports the conclusion that citrus fruit without leaves is not an epidemiologically significant pathway for the entry, establishment, and spread of P. citricarpa.

Ascosporic infection plays a major role in the epidemiology of citrus black spot (CBS) in South Africa, a disease caused by Phyllosticta citricarpa. Phyllosticta pseudothecium maturation and ascospore release models have been integrated in infection models to predict the availability of the primary inoculum source. However, these models have not been validated on a broader data set and this study aimed to validate and improve these epidemiological models. New pseudothecium maturation and ascospore release models for P. citricarpa were developed, based on weather and ascospore trap data from 13 locations and up to five seasons. From the 29 data sets analysed, 3775 3-hourly periods with ascospore events were recorded on 1798 days; 90% of these events occurred between 16.0 °C and 32.1 °C (daily Tmin and Tmax of 15.4 °C and 33.5 °C, respectively) and 75% occurred above a relative humidity (RH) of 55.9% (daily RH > 47.9%). Rain was recorded during 13.8% of these ascospore events and 20.0% of ascospore days. Using logistic regression, a Gompertz model that best predicted pseudothecium maturation, or the probability of onset of ascospore release, was developed and was markedly more accurate than the previously described models. The model consisted of DDtemp [cumulative degree-days from midwinter (1 July) calculated as (minimum + maximum daily temperature) / 2 – 10 °C] and DDwet (DDtemp accumulated only on days with >0.1 mm rain or vapour pressure deficit 0.1 mm rain or vapour pressure deficit

Citrus black spot (CBS), caused by

Huanglongbing (HLB, Asian Citrus Greening), the most devastating disease of citrus has not been detected in southern Africa (Gottwald, 2010). HLB is associated with 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium vectored by Diaphorina citri Kuwayama (Hemiptera: Liviidae), the Asian Citrus Psyllid (ACP). African Citrus Greening, associated with 'Candidatus Liberibacter africanus' (CLaf) and its vector the African Citrus Triozid, Trioza erytreae (Del Guercio) (Hemiptera: Triozidae), are endemic to Africa, although not previously reported from Angola. African Greening is less severe than HLB, largely due to heat sensitivity of CLaf and its vector. Introduction of HLB into southern Africa would be devastating to citrus production in commercial and informal sectors. Concern was raised that CLas or ACP might hae inadvertently been introduced into Angola. In July 2019, a survey was conducted in two citrus nurseries in Luanda and Caxito and in different orchards on 7 farms surrounding Calulo and Quibala. Yellow sticky traps for insects were placed at the various localities and collected after c. 3 weeks. Breeding signs of T. erytreae (pit galls) were observed on citrus in some locations, but no insect vectors were detected on traps. Trees were inspected for signs and symptoms of citrus pests and diseases, particularly those that resemble HLB (foliar blotchy mottle, shoot chlorosis, vein yellowing and corking, lopsided fruit with aborted seeds and colour inversion) and its vectors (pit galls on leaves or waxy exudates). Leaves and shoots with suspect symptoms were sampled for laboratory analysis (43 samples). DNA was extracted from petiole and midrib tissue of leaves using a modified CTAB extraction protocol of Doyle and Doyle (1990). Real-time PCR was done using universal Liberibacter primers of Roberts et al. (2015), CLaf specific primers of Li et al. (2006) and CLas specific primers of Bao et al. (2019). All real-time PCR protocols indicated the presence of CLaf in 6 samples (Tab. S1). CLas or other citrus Liberibacter species were not detected. The presence of CLaf in sample 37 was confirmed by constructing a library (NEXTFLEX® DNA Sequencing Kit, PerkinElmer) with extracted DNA and performing high-throughput sequencing on an Ion Torrent™ S5™ platform (Central Analytical Facility, Stellenbosch University). To improve the quality of the reads, all 233,617,700 obtained reads were trimmed from the 3' end to a maximum length of 240 nt using Trimmomatic (Bolger et al. 2014). The high quality reads were mapped to the Citrus sinensis reference genome (NC_023046.1) using Bowtie 2.3.4 (Langmead and Salzberg 2012) to subtract all the reads that had high identity to the host plant (number of mismatches allowed in the seed was set to 1). The 14,691,369 unmapped reads (6.2% of original data) were mapped to the CLaf reference genome NZ_CP004021.1 using CLC Genomics Workbench 10.1.1 (Qiagen) (Length fraction = 0.8; Similarity fraction = 0.9). A CLaf consensus genome was generated that spanned 99.7% of the reference genome and the 163001 mapped reads had a 22.9 mean read coverage. The consensus sequence was 99.7% identical to NZ_CP004021.1 and was submitted to Genbank as accession: CP054879. The positive CLaf detections were from trees with typical HLB or African Citrus Greening symptoms, viz. lopsided fruit with green stylar ends, aborted seed and stained columella at base of fruit button; yellow shoots with leaves showing symptoms of blotchy mottle and vein yellowing and corking (Fig. S1) in a commercial citrus farm outside Calulo and included 2 'Ponkan' mandarin (C. reticulata), 2 Valencia and 1 'Navelina' tree (C. sinensis), and a citrus nursery in Luanda (1 lime tree; C. aurantifolia) (Tab. S1). This first report of CLaf in Angola highlights the need to prevent spread by removing infected trees and managing the insect vector, as well as the need for further surveys to determine the occurrence of African Greening and its vectors in other provinces and to confirm the absence of exotic citrus pests and diseases. References Bao, M. et al. 2020. Plant Dis. 104:527 Bolger, A. M. et al. 2014. Bioinformatics. 30:2114-2120. Doyle, J.J. and Doyle, J.L. 1990. Focus 12:13 Gottwald, T.R. 2010. Annu. Rev. Phytopathol. 48:119 Langmead, B. and Salzberg, S. 2012. Nature Methods. 9:357-359. Li, W. et al. 2006. Jnl. Microbiol. Methods 66:104 Roberts, R. et al. 2015. Int. J. Syst. Evol. Micr. 65:723.

Background: Placentation (invasion of the placenta into the maternal endometrium) is hypothesised to be critical for healthy placental function and is abnormal in two thirds of miscarriage. Kisspeptin has emerged as a putative regulator of physiological placentation; it is highly expressed in placental syncitio-trophoblasts, whereas its receptor is expressed in both syncitio- and cyto-trophoblasts, such that kisspeptin is hypothesized to play an important paracrine role to regulate placentation. Circulating kisspeptin levels are considerably raised during healthy pregnancy and are reduced in women with miscarriage. Aim: We aimed to investigate the utility of circulating kisspeptin concentrations in the assessment of pregnancy complications and assess whether kisspeptin provides additional diagnostic information compared to beta human chorionic gonadotropin (βhCG) alone. Methods: This study was performed in collaboration with the Early Pregnancy Outcome Study (EPOS), which aims to identify novel pregnancy biomarkers. Women were invited to attend every fortnight for blood-sampling, clinical and ultrasound assessment during the first trimester, and repeated during the second and third trimesters. Asymptomatic women with healthy pregnancy (n=265) provided 960 blood-samples. Women with pregnancy complications including miscarriage (n=95), pre-eclampsia (PET; n=24), pregnancy induced hypertension (PIH; n=14), gestational diabetes (GDM; n=41), preterm birth (PTB; n=14) and intrauterine growth restriction (IUGR; n=24) provided 569 blood-samples. Results: Gestation-adjusted circulating kisspeptin and βhCG levels were lower, by 66% and 57%, respectively, in women with miscarriage compared to healthy pregnant controls (p Conclusions: Plasma kisspeptin is a promising biomarker for pregnancy complications and provides additional diagnostic capability over that provided by βhCG alone.

Citrus black spot (CBS) is caused by Phyllosticta citricarpa, which is regarded as a quarantine pathogen in certain countries. Pest risk assessments disagree on the risk of fruit as a pathway for introduction of the pest to new areas. Some countries accordingly regulate the movement of fruit from production regions where CBS occurs. Preharvest fungicide sprays are very effective in controlling CBS, but cannot consistently achieve complete control. The effect of postharvest treatments on CBS infections and on the reproductive ability of P. citricarpa in lesions that formed after these treatments was studied. Trials were conducted using naturally infected ‘Eureka’ lemon and Valencia orange fruit. Asymptomatic fruit were treated using commonly used packhouse sanitation and fungicide treatments, and cold storage (individually and combined), as well as alternative stand-alone treatments. After treatment, fruit were stored for 5 weeks (in the dark) at ambient temperature (20–22 °C) or −0.5–7 °C, whereafter they were incubated for a further 2 weeks at conditions conducive to expression of symptoms and formation of pycnidia. Individual treatments generally resulted in variable levels of control. The combination of packhouse treatments (including pre-packhouse drench with guazatine or propiconazole in combination with pyrimethanil, thiabendazole and 2,4-D; chlorine wash; dip treatment in imazalil; and brush application of a wax coating incorporated with imazalil, thiabendazole and 2,4-D) consistently showed moderate to high levels of control of CBS development from latent infections on lemons (32.4–43.4% for incidence and 61.6–68.3% for severity) and oranges (58.3–85.7% for incidence and 54.1%–88.8% for severity). However, cold storage subsequent to packhouse treatments (as is common shipping protocol) further improved the levels of control (44.0–58.3% for incidence and 78.1–82.5% for severity on lemons; 66.1–93.5% for incidence and 85.3–98.5% for severity on oranges). An average of 10% of new lesions on lemons and 15% on oranges formed pycnidia, indicating that P. citricarpa generally had a low reproductive capability in fruit lesions, which was in most cases further diminished by the combination of treatments followed by cold storage.

Summary Several Phyllosticta species are known as pathogens of Citrus spp., and are responsible for various disease symptoms including leaf and fruit spots. One of the most important species is P. citricarpa, which causes a foliar and fruit disease called citrus black spot. The Phyllosticta species occurring on citrus can most effectively be distinguished from P. citricarpa by means of multilocus DNA sequence data. Recent studies also demonstrated P. citricarpa to be heterothallic, and reported successful mating in the laboratory. Since the domestication of citrus, different clones of P. citricarpa have escaped Asia to other continents via trade routes, with obvious disease management consequences. This pathogen profile represents a comprehensive literature review of this pathogen and allied taxa associated with citrus, focusing on identification, distribution, genomics, epidemiology and disease management. This review also considers the knowledge emerging from seven genomes of Phyllosticta spp., demonstrating unknown aspects of these species, including their mating behaviour. Taxonomy Phyllosticta citricarpa (McAlpine) Aa, 1973. Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Botryosphaeriales, Family Phyllostictaceae, Genus Phyllosticta, Species citricarpa. Host range Confirmed on more than 12 Citrus species, Phyllosticta citricarpa has only been found on plant species in the Rutaceae. Disease symptoms P. citricarpa causes diverse symptoms such as hard spot, virulent spot, false melanose and freckle spot on fruit, and necrotic lesions on leaves and twigs. Useful websites DOE Joint Genome Institute MycoCosm portals for the Phyllosticta capitalensis (https://genome.jgi.doe.gov/Phycap1), P. citriasiana (https://genome.jgi.doe.gov/Phycit1), P. citribraziliensis (https://genome.jgi.doe.gov/Phcit1), P. citrichinaensis (https://genome.jgi.doe.gov/Phcitr1), P. citricarpa (https://genome.jgi.doe.gov/Phycitr1, https://genome.jgi.doe.gov/Phycpc1), P. paracitricarpa (https://genome.jgi.doe.gov/Phy27169) genomes. All available Phyllosticta genomes on MycoCosm can be viewed at https://genome.jgi.doe.gov/Phyllosticta.

Hepatitis C virus (HCV) infection induces interferon (IFN)-stimulated genes (ISGs) and downstream innate immune responses. This study investigated whether baseline and on-treatment differences in these responses predict response versus virological breakthrough during therapy with direct-acting antivirals (DAAs). Thirteen HCV genotype 1b-infected patients who had previously failed a course of pegylated IFN/ribavirin were retreated with asunaprevir/daclatasvir for 24 weeks. After pretreatment biopsy, patients were randomized to undergo a second biopsy at week 2 or 4 on therapy. Microarray and NanoString analyses were performed on paired liver biopsies and analyzed using linear mixed models. As biomarkers for peripheral IFN responses, peripheral blood natural killer cells were assessed for phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and degranulation. Nine of 13 (69%) patients achieved sustained virological response at 12 weeks off therapy (SVR12), and 4 experienced virological breakthroughs between weeks 4 and 12. Patients who achieved SVR12 displayed higher ISG expression levels in baseline liver biopsies and a higher frequency of pSTAT1 and TRAIL-expressing, degranulating natural killer cells in baseline blood samples than those who experienced virological breakthrough. Comparing gene expression levels from baseline and on-therapy biopsies, 408 genes (±1.2-fold, P < 0.01) were differentially expressed. Genes down-regulated on treatment were predominantly ISGs. Down-regulation of ISGs was rapid and correlated with HCV RNA suppression. Conclusion: An enhanced IFN signature is observed at baseline in liver and blood of patients who achieve SVR12 compared to those who experience a virological breakthrough; the findings suggest that innate immunity may contribute to clearance of HCV during DAA therapy by preventing the emergence of resistance-associated substitutions that lead to viral breakthrough during DAA therapy.

Elsinoe punicae was shown to be responsible for a new disease on pomegranates in South Africa. This disease has been found in three of the pomegranate production areas in the Cape provinces, and is characterised by scab-like symptoms on the fruit and brown spots on the leaves. Representative isolates were characterised by morphology, cultural growth and sequencing of the ITS. Additionally, the LSU, rpb2 and TEF1-α gene regions were also sequenced for one representative isolate from pomegranate. Phylogenetic analyses of the four loci confirmed the isolates as E. punicae. RAPD analyses were also performed with three primers on 19 isolates of E. punicae. The analyses showed that E. punicae isolates clustered together with high support apart from the reference isolates of E. australis, E. fawcetti and E. australis pathotype jojoba. Pathogenicity tests were conducted with E. punicae and two reference isolates (E. australis, E. fawcetti - Citrus pathogens) on whole plant leaves and detached leaves of pomegranate and different citrus types [grapefruit, rough lemon, navels, Valencias and mandarins] in official quarantine facilities. The results confirmed E. punicae as a pathogen of pomegranate and not citrus.

Mancozeb spray deposition and the persistence thereof to rainfall are important factors influencing the efficacy of apple scab control caused by Venturia inaequalis. Fungicide deposition can be assessed through quantification of fungicide residue, or fluorescent pigment quantification. Fluorescent pigment quantification is a more cost effective and less labour intensive method, since it is assessed using only photomacrography and image analyses. The yellow fluorescent pigment used in this study was shown to be a suitable tracer for five different mancozeb formulations on apple leaves when evaluated at different concentrations (0.5, 1.0, 1.5 and 2.0). Pearson's correlation was significant (P 0.495) between the three treatments based on FPC% and mancozeb residue quantity when simulated rain was applied to apple seedlings at a constant rainfall intensity of 5 mm/h at five different rainfall volumes (0, 1, 5, 10 and 15 mm). A fair to good correlation (r = 0.697 and 0.995) existed between FPC% and mancozeb residue, and their percentage loss at different rainfall volumes. However, the response of FPC% and mancozeb residue differed based on exponential regression curves. This was due to a markedly larger predicted loss by each model's asymptote, 51.67% for FPC% and 40.76% for mancozeb residue. Based on actual mancozeb residue values, a significant percentage loss in residue already occurred after applying 1 mm (32.90%) rain. When rain volumes were increased to 5 and 10 mm rain, the percentage losses (37.88 and 41.08%) did not differ significantly from 1 mm rain. The same was true for percentage loss in FPC%, except that a significant higher loss occurred at 10 mm (52.36%) than at 1 mm rain (41.13%).

Background Stress has demonstrated effects on inflammation though underlying cell-cell communication mechanisms remain unclear. We hypothesize that circulating RNAs and extracellular vesicles (EVs) in patients with chronic stress contain signals with functional roles in cell repair. Methods Blood transcriptome from patients with Irritable Bowel Syndrome versus controls were compared to identify signaling pathways and effectors. Plasma EVs were isolated (size-exclusion chromatography) and characterized for effectors' presence (immunogold labelling-electron microscopy). Based on transcriptome pathways and EV-labelling, lysozyme's effects on cell migration were tested in human colon epithelial CRL-1790 cells and compared to the effects of CXCL12, a migration inducer (wound assay). The effect of lysozyme on immune-linked mRNA and protein levels in cells which survived following serum starvation and scratch wound were investigated (NanoString). Results Blood transcriptomes revealed pyridoxal 5’phosphate salvage, pyrimidine ribonucleotides salvage pathways, atherosclerosis, and cell movement signaling with membrane CD9 and extracellular lysozyme as effectors. Plasma EVs showed labelling with CD9, mucins, and lysozyme. This is the first identification of lysozyme on plasma EVs. In CRL-1790 cells, lysozyme induced migration and repaired scratch wound as well as CXCL12. Immune mRNA and protein expressions were altered in cells which survived following serum starvation and scratch wound, with or without lysozyme in serum-free media post-wounding: CD9, IL8, IL6 mRNAs and CD9, NT5E, PD-L1 proteins. Conclusions Repair and inflammatory signals are identified in plasma EVs and circulating RNAs in chronic stress. Registered clinicaltrials.gov #NCT00824941 General significance This study highlights the role of circulating RNAs and EVs in stress., Highlights • Inflammatory pathways are predicted by blood transcriptome. • Plasma EVs from patients and controls show labelling with lysozyme, CD9, and mucins. • Lysozyme induces cell migration in response to wound in vitro. • Lysozyme alters immune-linked mRNA and protein cellular levels in response to wound. • Circulating RNAs and EVs contain signals with roles in cell migration and repair.

A total of 213 Penicillium digitatum isolates with resistance to the fungicide imazalil (IMZ) originating from green moulded citrus fruit from several countries (South Africa, USA, Uruguay, Spain, Israel, Cyprus, Chile, Australia and Argentina) were tested for their Cyp51 IMZ-resistance group (R1 to R3) using a published multiplex PCR assay. As well, the level of resistance (measured as EC50 values) was determined. Isolates from the USA showed the highest resistance group diversity (isolates belonged to R1, R2 and R3 groups) and lowest EC50 values. The isolates from the other countries were all classified into the R3 group, except for Chile, which also contained R1 isolates. There was no correlation between the level of resistance of isolates and the resistance group genotype. In general, IMZ resistance was most commonly associated (88.3%) with the R3 group. This study is the first to investigate the distribution of IMZ resistance genotypes in P. digitatum populations from a broad international scale.

Stress is known to perturb the microbiome and exacerbate irritable bowel syndrome (IBS) associated symptoms. Characterizing structural and functional changes in the microbiome is necessary to understand how alterations affect the biomolecular environment of the gut in IBS. Repeated water avoidance (WA) stress was used to induce IBS-like symptoms in rats. The colon-mucosa associated microbiome was characterized in 13 stressed and control animals by 16S sequencing. In silico analysis of the functional domains of microbial communities was done by inferring metagenomic profiles from 16S data. Microbial communities and functional profiles were compared between conditions. WA animals exhibited higher α-diversity and moderate divergence in community structure (β-diversity) compared with controls. Specific clades and taxa were consistently and significantly modified in the WA animals. The WA microbiome was particularly enriched in Proteobacteria and depleted in several beneficial taxa. A decreased capacity in metabolic domains, including energy- and lipid-metabolism, and an increased capacity for fatty acid and sulfur metabolism was inferred for the WA microbiome. The stressed condition favored the proliferation of a greater diversity of microbes that appear to be functionally similar, resulting in a functionally poorer microbiome with implications for epithelial health. Taxa, with known beneficial effects, were found to be depleted, which supports their relevance as therapeutic agents to restore microbial health. Microbial sulfur metabolism may form a key component of visceral nerve sensitization pathways and is therefore of interest as a target metabolic domain in microbial ecological restoration.

Abdominal pain is a chronic condition experienced by approximately 20% of individuals in the United States. The purpose of the study was to assess the validity of the Gastrointestinal Pain Pointer as a measure of abdominal pain intensity. A prospective longitudinal time-series study design was utilized. The sample included 93 outpatients (58.1% female). Participants met Rome III criteria for irritable bowel syndrome (n = 32) or were healthy controls (n = 61). The Gastrointestinal Pain Pointer, a new electronic pain assessment tool, was used to assess self-reported abdominal pain intensity among participants before and after ingestion of an intestinal permeability test solution across 11 time points over a 5-hour time period. The results were compared with the Short-Form McGill Pain Questionnaire. The Gastrointestinal Pain Pointer was found to be valid in the assessment of abdominal pain intensity. The tool is a novel and valid measure of abdominal pain intensity that enhances the ability for clinicians to better quantify, in real time, patient-related pain outcomes for both clinical care and research.

Colonic epithelial health is implicated in a host of gastrointestinal (GI) diseases and disorders. Lysozyme is suspected to play a role in the ability of the epithelium to recover from injury (Abey et al., in press; Gallo, 2012; Rubio, 2014) [1] , [2] , [3] . Disrupted repair mechanisms may lead to delayed or ineffective recovery and disruptions to epithelial biology resulting in GI symptoms and altered barrier function (Peterson and Artis, 2014) [4] . The effect of lysozyme on the transcriptomic and proteomic profile of healthy colonic epithelial cells was investigated. Epithelial cells in culture were scratch wounded and treated with lysozyme. mRNA and protein profiles were simultaneously quantified in the same sample using a digital counting technology. Gene and protein expressions altered by the presence or absence of lysozyme are described in this article. Extensive statistical and bioinformatic analysis, and interpretation of the results can be found in “Lysozyme association with circulating RNA, extracellular vesicles, and chronic stress” (Abey et al., in press) [1] .