Phytophthora root and collar rot of Paulownia, a new disease for Europe

Paulownia species are fast growing trees native to China, which are being grown in managed plantings in several European countries for the production of wood and biomasses. In 2018, wilting, stunting, leaf yellowing, and collapse, as a consequence of root and crown rot, were observed in around 40% of trees of a 2-year-old planting of Paulownia elongata × P. fortunei in Calabria (Southern Italy). Two species of Phytophthora were consistently recovered from roots, basal stem bark, and rhizosphere soil of symptomatic trees and were identified as Ph. nicotianae and Ph. palmivora on the basis of both morphological characteristics and phylogenetic analysis of rDNA ITS sequences. Koch’s postulates were fulfilled by reproducing the symptoms on potted paulownia saplings transplanted into infested soil or stem-inoculated by wounding. Both Phytophthora species were pathogenic and caused root rot and stem cankers. Even though P. palmivora was the only species recovered from roots of naturally infected plants, in pathogenicity tests through infested soil P. nicotianae was more virulent. This is the first report of Phytophthora root and crown rot of a Paulownia species in Europe. Strategies to prevent this emerging disease include the use of healthy nursery plants, choice of well-drained soils for new plantations, and proper irrigation management

Characterization of alternaria species associated with heart rot of pomegranate fruit

This study was aimed at identifying Alternaria species associated with heart rot disease of pomegranate fruit in southern Italy and characterizing their mycotoxigenic profile. A total of 42 Alternaria isolates were characterized. They were obtained from pomegranate fruits with symptoms of heart rot sampled in Apulia and Sicily and grouped into six distinct morphotypes based on macro-and microscopic features. According to multigene phylogenetic analysis, including internal transcribed spacer (ITS), translation elongation factor 1-α (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a SCAR marker (OPA10-2), 38 isolates of morphotypes 1 to 5 were identified as Alternaria alternata, while isolates of morphotype 6, all from Sicily, clustered within the Alternaria arborescens species complex. In particular, isolates of morphotype 1, the most numerous, clustered with the ex-type isolate of A. alternata, proving to belong to A. alternata. No difference in pathogenicity on pomegranate fruits was found between isolates of A. alternata and A. arborescens and among A. alternata isolates of different morphotypes. The toxigenic profile of isolates varied greatly: in vitro, all 42 isolates produced tenuazonic acid and most of them other mycotoxins, including alternariol, alternariol monomethyl ether, altenuene and tentoxin

A new approach in the monitoring of the phytosanitary conditions of forests: the case of oak and beech stands in the Sicilian Regional Parks

L'obiettivo del presente studio è stato quello di esaminare lo stato fitosanitario delle quercete e faggete dei tre Parchi Regionali Siciliani (Parco dell'Etna, Parco delle Madonie, Parco dei Nebrodi). Lo studio è stato condotto individuando delle aree di saggio, popolamenti forestali omogenei sotto l'aspetto floristico, ecologico e fitosanitario. Complessivamente sono state delimitate 81 aree di saggio, di cui 54 quercete e 27 faggete. La condizione fitosanitaria di ogni essenza arborea all'interno della rispettiva area di saggio è stata espressa con un indice numerico denominato “classe fitosanitaria” (PC). I popolamenti di quercia hanno mostrato un elevato grado di sofferenza, con alberi sintomatici nell’85% delle aree di saggio. I popolamenti di faggio hanno mostrato una situazione di maggiore stabilità, ad eccezione delle faggete del parco dei Nebrodi che apparivano molto degradate. Sul genere Quercus, sono stati osservati sintomi di infezioni di patogeni fungini comuni nelle foreste delle aree temperate e Mediterranee, quali Biscogniauxia mediterranea, Polyporus sp., Fistulina hepatica, Mycrosphaera alphitoides ed Armillaria sp., mentre su faggio sono state osservate infezioni di Biscogniauxia nummularia, Fomes fomentarius e Neonectria radicicola. Sono state altresì individuate 22 aree che vengono proposte come aree di saggio permanenti dello stato fitosanitario delle foreste nei tre parchi.The objective of this study was to investigate the health conditions of oak and beech stands in the three Regional Parks of Sicily (Etna, Madonie and Nebrodi). A total of 81 sampling areas were investigated, 54 in oak stands and 27 in beech stands. The phytosanitary conditions of each tree within the respective sampling area was expressed with a synthetic index namely phytosanitary class (PC). Oak stands showed severe symptoms of decline, with 85% of the sampling areas including symptomatic trees. In general, beech stands were in better condition, with the exception of Nebrodi Park, where trees showed severe symptoms of decline. On oak trees, infections of fungal pathogens were also observed, including Biscogniauxia mediterranea, Polyporus sp., Fistulina hepatica, Mycrosphaera alphitoides and Armillaria sp. By contrast, on beech trees Biscogniauxia nummularia, Fomes fomentarius and Neonectria radicicola were recognized. Furthermore, twenty-two permanent sampling areas were delimited with the aim of monitoring regularly the health conditions of forests in these three parks

Protocol for a multinational risk-stratified randomised controlled trial in paediatric Crohn's disease: methotrexate versus azathioprine or adalimumab for maintaining remission in patients at low or high risk for aggressive disease course

INTRODUCTION: Immunomodulators such as thiopurines (azathioprine (AZA)/6-mercaptopurine (6MP)), methotrexate (MTX) and biologics such as adalimumab (ADA) are well established for maintenance of remission within paediatric Crohn's disease (CD). It remains unclear, however, which maintenance medication should be used first line in specific patient groups. AIMS: To compare the efficacy of maintenance therapies in newly diagnosed CD based on stratification into high and low-risk groups for severe CD evolution; MTX versus AZA/6MP in low-risk and MTX versus ADA in high-risk patients. Primary end point: sustained remission at 12 months (weighted paediatric CD activity index ≤12.5 and C reactive protein ≤1.5 fold upper limit) without relapse or ongoing requirement for exclusive enteral nutrition (EEN)/steroids 12 weeks after treatment initiation. METHODS AND ANALYSIS: REDUCE-RISK in CD is an international multicentre open-label prospective randomised controlled trial funded by EU within the Horizon2020 framework (grant number 668023). Eligible patients (aged 6-17 years, new-onset disease receiving steroids or EEN for induction of remission for luminal ± perianal CD are stratified into low and high-risk groups based on phenotype and response to induction therapy. Participants are randomised to one of two treatment arms within their risk group: low-risk patients to weekly subcutaneous MTX or daily oral AZA/6MP, and high-risk patients to weekly subcutaneous MTX or fortnightly ADA. Patients are followed up for 12 months at prespecified intervals. Electronic case report forms are completed prospectively. The study aims to recruit 312 participants (176 low risk; 136 high risk). ETHICS AND DISSEMINATION: ClinicalTrials.gov Identifier: (NCT02852694), authorisation and approval from local ethics committees have been obtained prior to recruitment. Individual informed consent will be obtained prior to participation in the study. Results will be published in a peer-reviewed journal with open access. TRIAL REGISTRATION NUMBER: NCT02852694; Pre-results

International prospective observational study investigating the disease course and heterogeneity of paediatric-onset inflammatory bowel disease: the protocol of the PIBD-SETQuality inception cohort study

INTRODUCTION: Patients with paediatric-onset inflammatory bowel disease (PIBD) may develop a complicated disease course, including growth failure, bowel resection at young age and treatment-related adverse events, all of which can have significant and lasting effects on the patient's development and quality of life. Unfortunately, we are still not able to fully explain the heterogeneity between patients and their disease course and predict which patients will respond to certain therapies or are most at risk of developing a more complicated disease course. To investigate this, large prospective studies with long-term follow-up are needed. Currently, no such European or Asian international cohorts exist. In this international cohort, we aim to evaluate disease course and which patients are most at risk of therapy non-response or development of complicated disease based on patient and disease characteristics, immune pathology and environmental and socioeconomic factors. METHODS AND ANALYSIS: In this international prospective observational study, which is part of the PIBD Network for Safety, Efficacy, Treatment and Quality improvement of care (PIBD-SETQuality), children diagnosed with inflammatory bowel disease <18 years are included at diagnosis. The follow-up schedule is in line with standard PIBD care and is intended to continue up to 20 years. Patient and disease characteristics, as well as results of investigations, are collected at baseline and during follow-up. In addition, environmental factors are being assessed (eg, parent's smoking behaviour, dietary factors and antibiotic use). In specific centres with the ability to perform extensive immunological analyses, blood samples and intestinal biopsies are being collected and analysed (flow cytometry, plasma proteomics, mRNA expression and immunohistochemistry) in therapy-naïve patients and during follow-up. ETHICS AND DISSEMINATION: Medical ethical approval has been obtained prior to patient recruitment for all sites. The results will be disseminated through peer-reviewed scientific publications. TRIAL REGISTRATION NUMBER: NCT03571373

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum

[EN] Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.This work was supported by COST action FP1406 Pinestrength . The work of the Estonian team was supported by the Estonian Science Foundation grants PSG136 and IUT21-04. The work of Portuguese team from INIAV was financed by INIAV I.P. Institute. The work at U. Aveiro (Portugal) was financed by European Funds through COMPETE and National Funds through the Portuguese Foundation for Science and Technology (FCT) to CESAM (UID/AMB/50017/2013 POCI-01- 0145-FEDER-007638). The work of Slovenian team was financed through Slovenian Research Agency (P4-0107) and by the Slovenian Ministry of Agriculture, Forestry and Food (Public Forestry Service). The British work was financially supported by the Forestry Commission, UK. The French work was financially supported by the French Agency for Food, environmental and occupational health safety (ANSES). The work in New Zealand was funded by Operational Research Programmes, Ministry for Primary Industries, New Zealand.Ioos, R.; Aloi, F.; Piskur, B.; Guinet, C.; Mullett, M.; Berbegal Martinez, M.; Bragança, H.... (2019). Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum. Scientific Reports. 9:1-17. https://doi.org/10.1038/s41598-019-44672-8S1179Schmale, D. G. III & Gordon, T. R. Variation in susceptibility to pitch canker disease, caused by Fusarium circinatum, in native stands of Pinus muricata. Plant Pathol. 52, 720–725 (2003).Gordon, T. R., Kirkpatrick, S. C., Aegerter, B. J., Wood, D. L. & Storer, A. J. Susceptibility of Douglas fir (Pseudotsuga menziesii) to pitch canker, caused by Gibberella circinata (anamorph = Fusarium circinatum). Plant Pathol. 55, 231–237 (2006).Martínez‐Álvarez, P., Pando, V. & Diez, J. J. Alternative species to replace Monterey pine plantations affected by pitch canker caused by Fusarium circinatum in northern Spain. Plant Pathol. 63, 1086–1094, https://doi.org/10.1111/ppa.12187 (2014).Wingfield, M. J. et al. Pitch canker caused by Fusarium circinatum - a growing threat to pine plantations and forests worldwide. Australas. Plant Path. 37, 319–334 (2008).Bezos, D., Martinez-Alvarez, P., Fernandez, M. & Diez, J. J. Epidemiology and management of pine pitch canker disease in Europe - a review. Balt. For. 23, 279–293 (2017).Landeras, E. et al. Outbreak of pitch canker caused by Fusarium circinatum on Pinus spp. in Northern Spain. Plant Dis. 89, 1015 (2005).Bragança, H., Diogo, E., Moniz, F. & Amaro, P. First report of pitch canker on pines caused by Fusarium circinatum in Portugal. Plant Dis. 93, 1079–1079, https://doi.org/10.1094/PDIS-93-10-1079A (2009).EFSA. Risk assessment of Gibberella circinata for the EU territory and identification and evaluation of risk management options. EFSA Journal 8, 1620 (2010).Carlucci, A., Colatruglio, L. & Frisullo, S. First report of pitch canker caused by Fusarium circinatum on Pinus halepensis and P. pinea in Apulia (Southern Italy). Plant Dis. 91, 1683 (2007).Vettraino, A., Potting, R. & Raposo, R. EU legislation on forest plant health: an overview with a focus on Fusarium circinatum. Forests 9, 568 (2018).Möykkynen, T., Capretti, P. & Pukkala, T. Modelling the potential spread of Fusarium circinatum, the causal agent of pitch canker in Europe. Annals of Forest Sciences 72, 169–181 (2015).Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55, https://doi.org/10.1373/clinchem.2008.112797 (2009).EPPO. PM 7/91(1): Gibberella circinata. EPPO Bull. 39, 298–309 (2009).ISTA. 7-009: Detection of Gibberella circinata on Pinus spp. (pine) and Pseudotsuga menziesii (Douglas-fir) seed. Validated Seed Health Testing Methods (2015).IPPC. ISPM 27, Diagnostic protocols for regulated pests, DP 22: Fusarium circinatum (2017).EPPO. PM 7/98 (2) Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. EPPO Bull. 44, 117–147, https://doi.org/10.1111/epp.12118 (2014).Nirenberg, H. I. & O’Donnell, K. New Fusarium species and combinations within the Gibberella fujikuroi species complex. Mycologia 90, 434–458 (1998).Britz, H., Coutinho, T. A., Wingfield, M. J. & Marasas, W. F. O. Validation of the description of Gibberella circinata and morphological differentiation of the anamorph Fusarium circinatum. Sydowia 54, 9–22 (2002).Mullett, M., Pérez-Sierra, A., Armengol, J. & Berbegal, M. Phenotypical and molecular characterisation of Fusarium circinatum: correlation with virulence and fungicide sensitivity. Forests 8, 458 (2017).Herron, D. A. et al. Novel taxa in the Fusarium fujikuroi species complex from Pinus spp. Stud. Mycol. 80, 131–150, https://doi.org/10.1016/j.simyco.2014.12.001 (2015).Storer, G. & Clark, S. L. Association of the pitch canker fungus, Fusarium subglutinans f.sp. pini, with Monterey pine seeds and seedlings in California. Plant Pathol. 47, 649–656, https://doi.org/10.1046/j.1365-3059.1998.00288.x (1998).Schweigkofler, W., O’Donnell, K. & Garbelotto, M. Detection and quantification of airborne conidia of Fusarium circinatum, the causal agent of pine pitch canker, from two California sites by using a real-time PCR approach combined with a simple spore trapping method. Appl. Environ. Microbiol. 70, 3512–3520 (2004).Ramsfield, T. D., Dobbie, K., Dick, M. A. & Ball, R. D. Polymerase chain reaction-based detection of Fusarium circinatum, the causal agent of pitch canker disease. Molecular Ecology Resources 8, 1270–1273 (2008).Ioos, R., Fourrier, C., Iancu, G. & Gordon, T. R. Sensitive Detection of Fusarium circinatum in Pine Seed by Combining an Enrichment Procedure with a Real-Time Polymerase Chain Reaction Using Dual-Labeled Probe Chemistry. Phytopathology 99, 582–590, https://doi.org/10.1094/PHYTO-99-5-0582 (2009).Dreaden, T. J., Smith, J. A., Barnard, E. L. & Blakeslee, G. Development and evaluation of a real-time PCR seed lot screening method for Fusarium circinatum, causal agent of pitch canker disease. For. Path. 42, 405–411, https://doi.org/10.1111/j.1439-0329.2012.00774.x (2012).Fourie, G. et al. Culture-independent detection and quantification of Fusarium circinatum in a pine-producing seedling nursery. Southern Forests: a Journal of Forest Science 76, 137–143, https://doi.org/10.2989/20702620.2014.899058 (2014).Lamarche, J. et al. Molecular detection of 10 of the most unwanted alien forest pathogens in Canada using Real-Time PCR. PLoS ONE 10, e0134265, https://doi.org/10.1371/journal.pone.0134265 (2015).Luchi, N., Pepori, A. L., Bartolini, P., Ioos, R. & Santini, A. Duplex real-time PCR assay for the simultaneous detection of Caliciopsis pinea and Fusarium circinatum in pine samples. Applied Microbiology and Biotechnology 102, 7135–7146, https://doi.org/10.1007/s00253-018-9184-1 (2018).Sandoval-Denis, M., Swart, W. J. & Crous, P. W. New Fusarium species from the Kruger National Park, South Africa. MycoKeys 34, https://doi.org/10.3897/mycokeys.34.25974 (2018).Steenkamp, E. T., Wingfield, B. D., Desjardins, A. E., Marasas, W. F. & Wingfield, M. J. Cryptic speciation in Fusarium subglutinans. Mycologia 94, 1032–1043 (2002).Garcia-Benitez, C. et al. Proficiency of real-time PCR detection of latent Monilinia spp. infection in nectarine flowers and fruit. Phytopathologia Mediterranea 56, 242–250 (2017).Ebentier, D. L. et al. Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods. Water Research 47, 6839–6848, https://doi.org/10.1016/j.watres.2013.01.060 (2013).Bustin, S. & Huggett, J. qPCR primer design revisited. Biomolecular Detection and Quantification 14, 19–28, https://doi.org/10.1016/j.bdq.2017.11.001 (2017).Grosdidier, M., Aguayo, J., Marçais, B. & Ioos, R. Detection of plant pathogens using real-time PCR: how reliable are late Ct values? Plant Pathol. 66, 359–367, https://doi.org/10.1111/ppa.12591 (2017).Al-Soud, W. A. & Rådström, P. Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Applied and environmental microbiology 64, 3748–3753 (1998).Saunders, G. C., Dukes, J., Parkes, H. C. & Cornett, J. H. Interlaboratory study on thermal cycler performance in controlled PCR and random amplified polymorphic DNA analyses. Clinical chemistry 47, 47–55 (2001).Boutigny, A.-L. et al. Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae. Fungal Biology 117, 389–398, https://doi.org/10.1016/j.funbio.2013.04.001 (2013).Guinet, C., Fourrier-Jeandel, C., Cerf-Wendling, I. & Ioos, R. One-step detection of Monilinia fructicola, M. fructigena, and M. laxa on Prunus and Malus by a multiplex real-time PCR assay. Plant Dis. 100, 2465–2474, https://doi.org/10.1094/PDIS-05-16-0655-RE (2016).Aguayo, J. et al. Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4. PLoS ONE 12, e0171767, https://doi.org/10.1371/journal.pone.0171767 (2017).Broeders, S. et al. Guidelines for validation of qualitative real-time PCR methods. Trends in Food Science & Technology 37, 115–126, https://doi.org/10.1016/j.tifs.2014.03.008 (2014).Pelloux, H. et al. A second European collaborative study on polymerase chain reaction for Toxoplasma gondii, involving 15 teams. FEMS Microbiology Letters 165, 231–237, https://doi.org/10.1111/j.1574-6968.1998.tb13151.x (1998).Leslie, J. F. & Summerell, B. A. The Fusarium laboratory manual. (Blackwell Publishing, 2006).Ioos, R. et al. Test performance study of diagnostic procedures for identification and detection of Gibberella circinata in pine seeds in the framework of a EUPHRESCO project. EPPO Bull. 43, 267–275, https://doi.org/10.1111/epp.12037 (2013).Geiser, D. M. FUSARIUM-ID v. 1.0: a DNA sequence database for identifying Fusarium. Eur. J. Plant Pathol. 110, 473–479 (2004).White, T. J., Bruns, T., Lee, S. & Taylor, J. In PCR protocols: a guide to method and applications (eds Gelfand, D. H., Innis M. A., Sninsky, J. J. and White, T. J.) 315–322 (Academic Press, 1990).Nirenberg, H. I. A simplified method for identifying Fusarium spp. occurring on wheat. Canadian Journal of Botany 59, 1599–1609 (1981).Chabirand, A., Loiseau, M., Renaudin, I. & Poliakoff, F. Data processing of qualitative results from an interlaboratory comparison for the detection of “Flavescence dorée” phytoplasma: How the use of statistics can improve the reliability of the method validation process in plant pathology. PLoS ONE 12, e0175247, https://doi.org/10.1371/journal.pone.0175247 (2017).Loreti, S. et al. Performance of diagnostic tests for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) from woody samples. European Journal of Plant Pathology, https://doi.org/10.1007/s10658-018-1509-5 (2018).International Standardization Organization. ISO 16140:2003 Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods (2003).Langton, S., Chevennement, R., Nagelkerke, N. & Lombard, B. Analysing collaborative trials for qualitative microbiological methods: accordance and concordance. International Journal of Food Microbiology 79, 175–181 (2002).R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna (2014). R Foundation for Statistical Computing (2017).Wickham, H. ggplot2 : elegant graphics for data analysis. (Springer, 2016)

Predicting outcomes in pediatric ulcerative colitis for management optimization: systematic review and consensus statements from the pediatric inflammatory bowel disease–ahead program

Background & Aims: A better understanding of prognostic factors in ulcerative colitis (UC) could improve patient management and reduce complications. We aimed to identify evidence-based predictors for outcomes in pediatric UC, which may be used to optimize treatment algorithms. Methods: Potential outcomes worthy of prediction in UC were determined by surveying 202 experts in pediatric UC. A systematic review of the literature, with selected meta-analysis, was performed to identify studies that investigated predictors for these outcomes. Multiple national and international meetings were held to reach consensus on evidence-based statements. Results: Consensus was reached on 31 statements regarding predictors of colectomy, acute severe colitis (ASC), chronically active pediatric UC, cancer and mortality. At diagnosis, disease extent (6 studies, N = 627; P =.035), Pediatric Ulcerative Colitis Activity Index score (4 studies, n = 318; P <.001), hemoglobin, hematocrit, and albumin may predict colectomy. In addition, family history of UC (2 studies, n = 557; P =.0004), extraintestinal manifestations (4 studies, n = 526; P =.048), and disease extension over time may predict colectomy, whereas primary sclerosing cholangitis (PSC) may be protective. Acute severe colitis may be predicted by disease severity at onset and hypoalbuminemia. Higher Pediatric Ulcerative Colitis Activity Index score and C-reactive protein on days 3 and 5 of hospital admission predict failure of intravenous steroids. Risk factors for malignancy included concomitant diagnosis of primary sclerosing cholangitis, longstanding colitis (>10 years), male sex, and younger age at diagnosis. Conclusions: These evidence-based consensus statements offer predictions to be considered for a personalized medicine approach in treating pediatric UC