Is the Emergence of Dothistroma Needle Blight of Pine in France Caused by the Cryptic Species Dothistroma pini?

International audienceDothistroma needle blight (DNB) emerged in France in the past 15 years. This disease is induced by two closely related species Dothistroma septosporum and D. pini. While both species are nowadays present in France, only D. septosporum was reported in the past. We investigated whether a recent arrival of D. pini in France could be a cause of the DNB emergence. We analysed herbarium specimens of pine needle with DNB symptoms using PCR techniques to study the past frequency of D. pini in France. We also determined the present distribution within the country of D. septosporum and D. pini and compared it to the spatial pattern of DNB report in the DSF data base (french forest health monitoring agency). Although D. pini was detected on herbarium specimens from 1907 and 1965, it was not frequent in France in the past. It is nowadays frequent although not present throughout the country, being absent from the north and the east. There is no relationship between the D. pini distribution in France and the spatial pattern of DNB report in the DSF database. Thus, the emergence of DNB in France cannot be explained by a recent arrival of D. pini

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)

Species Tree Estimation for the Late Blight Pathogen, Phytophthora infestans, and Close Relatives

To better understand the evolutionary history of a group of organisms, an accurate estimate of the species phylogeny must be known. Traditionally, gene trees have served as a proxy for the species tree, although it was acknowledged early on that these trees represented different evolutionary processes. Discordances among gene trees and between the gene trees and the species tree are also expected in closely related species that have rapidly diverged, due to processes such as the incomplete sorting of ancestral polymorphisms. Recently, methods have been developed for the explicit estimation of species trees, using information from multilocus gene trees while accommodating heterogeneity among them. Here we have used three distinct approaches to estimate the species tree for five Phytophthora pathogens, including P. infestans, the causal agent of late blight disease in potato and tomato. Our concatenation-based “supergene” approach was unable to resolve relationships even with data from both the nuclear and mitochondrial genomes, and from multiple isolates per species. Our multispecies coalescent approach using both Bayesian and maximum likelihood methods was able to estimate a moderately supported species tree showing a close relationship among P. infestans, P. andina, and P. ipomoeae. The topology of the species tree was also identical to the dominant phylogenetic history estimated in our third approach, Bayesian concordance analysis. Our results support previous suggestions that P. andina is a hybrid species, with P. infestans representing one parental lineage. The other parental lineage is not known, but represents an independent evolutionary lineage more closely related to P. ipomoeae. While all five species likely originated in the New World, further study is needed to determine when and under what conditions this hybridization event may have occurred

Global Geographic Distribution and Host Range of Fusarium circinatum, the Causal Agent of Pine Pitch Canker

Fusarium circinatum, the causal agent of pine pitch canker (PPC), is currently one of the most important threats of Pinus spp. globally. This pathogen is known in many pine-growing regions, including natural and planted forests, and can affect all life stages of trees, from emerging seedlings to mature trees. Despite the importance of PPC, the global distribution of F. circinatum is poorly documented, and this problem is also true of the hosts within countries that are affected. The aim of this study was to review the global distribution of F. circinatum, with a particular focus on Europe. We considered (1) the current and historical pathogen records, both positive and negative, based on confirmed reports from Europe and globally; (2) the genetic diversity and population structure of the pathogen; (3) the current distribution of PPC in Europe, comparing published models of predicted disease distribution; and (4) host susceptibility by reviewing literature and generating a comprehensive list of known hosts for the fungus. These data were collated from 41 countries and used to compile a specially constructed geo-database. A review of 6297 observation records showed that F. circinatum and the symptoms it causes on conifers occurred in 14 countries, including four in Europe, and is absent in 28 countries. Field observations and experimental data from 138 host species revealed 106 susceptible host species including 85 Pinus species, 6 non-pine tree species and 15 grass and herb species. Our data confirm that susceptibility to F. circinatum varies between different host species, tree ages and environmental characteristics. Knowledge on the geographic distribution, host range and the relative susceptibility of different hosts is essential for disease management, mitigation and containment strategies. The findings reported in this review will support countries that are currently free of F. circinatum in implementing effective procedures and restrictions and prevent further spread of the pathogen

Febrile seizures and mechanisms of epileptogenesis: insights from an animal model.

Temporal lobe epilepsy (TLE) is the most prevalent type of human epilepsy, yet the causes for its development, and the processes involved, are not known. Most individuals with TLE do not have a family history, suggesting that this limbic epilepsy is a consequence of acquired rather than genetic causes. Among suspected etiologies, febrile seizures have frequently been cited. This is due to the fact that retrospective analyses of adults with TLE have demonstrated a high prevalence (20-->60%) of a history of prolonged febrile seizures during early childhood, suggesting an etiological role for these seizures in the development of TLE. Specifically, neuronal damage induced by febrile seizures has been suggested as a mechanism for the development of mesial temporal sclerosis, the pathological hallmark of TLE. However, the statistical correlation between febrile seizures and TLE does not necessarily indicate a causal relationship. For example, preexisting (genetic or acquired) 'causes' that result independently in febrile seizures and in TLE would also result in tight statistical correlation. For obvious reasons, complex febrile seizures cannot be induced in the human, and studies of their mechanisms and of their consequences on brain molecules and circuits are severely limited. Therefore, an animal model was designed to study these seizures. The model reproduces the fundamental key elements of the human condition: the age specificity, the physiological temperatures seen in fevers of children, the length of the seizures and their lack of immediate morbidity. Neuroanatomical, molecular and functional methods have been used in this model to determine the consequences of prolonged febrile seizures on the survival and integrity of neurons, and on hyperexcitability in the hippocampal-limbic network. Experimental prolonged febrile seizures did not lead to death of any of the seizure-vulnerable populations in hippocampus, and the rate of neurogenesis was also unchanged. Neuronal function was altered sufficiently to promote synaptic reorganization of granule cells, and transient and long-term alterations in the expression of specific genes were observed. The contribution of these consequences of febrile seizures to the epileptogenic process is discussed

Sampling and Detection Strategies for the Pine Pitch Canker (PPC) Disease Pathogen Fusarium circinatum in Europe

Fusarium circinatum Nirenberg & O’Donnel is listed among the species recommended for regulation as quarantine pests in Europe. Over 60 Pinus species are susceptible to the pathogen and it also causes disease on Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and species in genera such as Picea and Larix. The European Food Safety Authority considers the probability of new introductions—via contaminated seeds, wood material, soil and growing substrates, natural means and human activities—into the EU very likely. Due to early detection, constant surveillance and control measures, F. circinatum outbreaks have officially been eradicated in Italy and France. However, the global spread of F. circinatum suggests that the pathogen will continue to be encountered in new environments in the future. Therefore, continuous surveillance of reproductive material, nurseries and plantations, prompt control measures and realistic contingency plans will be important in Europe and elsewhere to limit disease spread and the “bridgehead effect”, where new introductions of a tree pathogen become increasingly likely as new environments are invaded, must be considered. Therefore, survey programs already implemented to limit the spread in Europe and that could be helpful for other EU countries are summarized in this review. These surveys include not only countries where pitch canker is present, such as Portugal and Spain, but also several other EU countries where F. circinatum is not present. Sampling protocols for seeds, seedlings, twigs, branches, shoots, soil samples, spore traps and insects from different studies are collated and compiled in this review. Likewise, methodology for morphological and molecular identification is herein presented. These include conventional PCR with a target-specific region located in the intergenic spacer region, as well as several real-time PCR protocols, with different levels of specificity and sensitivity. Finally, the global situation and future perspectives are addressed

The Plant Pathogen Phytophthora andina Emerged via Hybridization of an Unknown Phytophthora Species and the Irish Potato Famine Pathogen, P. infestans

Emerging plant pathogens have largely been a consequence of the movement of pathogens to new geographic regions. Another documented mechanism for the emergence of plant pathogens is hybridization between individuals of different species or subspecies, which may allow rapid evolution and adaptation to new hosts or environments. Hybrid plant pathogens have traditionally been difficult to detect or confirm, but the increasing ease of cloning and sequencing PCR products now makes the identification of species that consistently have genes or alleles with phylogenetically divergent origins relatively straightforward. We investigated the genetic origin of Phytophthora andina, an increasingly common pathogen of Andean crops Solanum betaceum, S. muricatum, S. quitoense, and several wild Solanum spp. It has been hypothesized that P. andina is a hybrid between the potato late blight pathogen P. infestans and another Phytophthora species. We tested this hypothesis by cloning four nuclear loci to obtain haplotypes and using these loci to infer the phylogenetic relationships of P. andina to P. infestans and other related species. Sequencing of cloned PCR products in every case revealed two distinct haplotypes for each locus in P. andina, such that each isolate had one allele derived from a P. infestans parent and a second divergent allele derived from an unknown species that is closely related but distinct from P. infestans, P. mirabilis, and P. ipomoeae. To the best of our knowledge, the unknown parent has not yet been collected. We also observed sequence polymorphism among P. andina isolates at three of the four loci, many of which segregate between previously described P. andina clonal lineages. These results provide strong support that P. andina emerged via hybridization between P. infestans and another unknown Phytophthora species also belonging to Phytophthora clade 1c