Effect of gaseous ozone treatment on the aroma and clove rot by Fusarium proliferatum during garlic postharvest storage

It is known that garlic bulbs preserved with traditional methods undergo considerable losses, ranging from 25 to 40%. A frequent cause of these losses is associated with the development of pathogenic fungi, such as those of the genus Fusarium. The effect of ozone on post-harvest garlic bulbs was evaluated. Garlic cloves inoculated with Fusarium proliferatum F21 and F22 strains, were exposed to a continuous gaseous ozone flow (2.14 μg m−3), during 4 days, 20 h a day. After ozone-treatment, the garlic samples were moved at 22 °C to mimic retail conditions (shelf life). The changes in several quality parameters such as fungal decay and aroma were evaluated on garlic samples, as whole bulbs, cloves with and without tunic, through a sensorial descriptive test, SPME analysis in GC/MS and microbiological approaches. The data collected showed that ozone treatment did not affect the aromatic profile of garlic. A significant detrimental effect of ozone treatment on garlic decay was observed. Our results encourage the use of gaseous ozone treatment for containing garlic fungal decay during its storage

Commodity risk assessment of black pine (Pinus thunbergii Parl.) bonsai from Japan

The EFSA Panel on Plant health was requested to deliver a scientific opinion on how far the existing requirements for the bonsai pine species subject to derogation in Commission Decision 2002/887/EC would cover all plant health risks from black pine (Pinus thunbergii Parl.) bonsai (the commodity defined in the EU legislation as naturally or artificially dwarfed plants) imported from Japan, taking into account the available scientific information, including the technical information provided by Japan. The relevance of an EU-regulated pest for this opinion was based on: (a) evidence of the presence of the pest in Japan; (b) evidence that P.\ua0thunbergii is a host of the pest and (c) evidence that the pest can be associated with the commodity. Sixteen pests that fulfilled all three criteria were selected for further evaluation. The relevance of other pests present in Japan (not regulated in the EU) for this opinion was based on (i) evidence of the absence of the pest in the EU; (ii) evidence that P.\ua0thunbergii is a host of the pest; (iii) evidence that the pest can be associated with the commodity and (iv) evidence that the pest may have an impact in the EU. Three pests fulfilled all four criteria and were selected for further evaluation (Crisicoccus pini, Sirex nitobei and Urocerus japonicus). For the selected 19 pests, the risk mitigation measures proposed in the technical dossier were evaluated. Limiting factors on the effectiveness of the measures were documented. For each of the 19 pests, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including any uncertainties. For all evaluated pests, the median likelihood of the pest freedom is 99.5% or higher and within the 90% uncertainty range it is 99% or higher

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)

Distribution and diversity of Phytophthora across Australia

The introduction and subsequent impact of Phytophthora cinnamomi within native vegetation is one of the major conservation issues for biodiversity in Australia. Recently, many new Phytophthora species have been described from Australia's native ecosystems; however, their distribution, origin, and potential impact remain unknown. Historical bias in Phytophthora detection has been towards sites showing symptoms of disease, and traditional isolation methods show variable effectiveness of detecting different Phytophthora species. However, we now have at our disposal new techniques based on the sampling of environmental DNA and metabarcoding through the use of high-throughput sequencing. Here, we report on the diversity and distribution of Phytophthora in Australia using metabarcoding of 640 soil samples and we compare the diversity detected using this technique with that available in curated databases. Phytophthora was detected in 65% of sites, and phylogenetic analysis revealed 68 distinct Phytophthora phylotypes. Of these, 21 were identified as potentially unique taxa and 25 were new detections in natural areas and/or new introductions to Australia. There are 66Phytophthora taxa listed in Australian databases, 43 of which were also detected in this metabarcoding study. This study revealed high Phytophthora richness within native vegetation and the additional records provide a valuable baseline resource for future studies. Many of the Phytophthora species now uncovered in Australia's native ecosystems are newly described and until more is known we need to be cautious with regard to the spread and conservation management of these new species in Australia's unique ecosystems