Genetic diversity and virulence variability in Diplodia mutila isolates from symptomatic grapevines in New Zealand: Virulence and genetic diversity of Diplodia mutila

Genetic diversity and virulence variability of Diplodia mutila isolates recovered from grapevines in New Zealand were investigated. The universally primed PCR (UP-PCR) and vegetative compatibility group (VCG) methods were used to investigate the genetic diversity. Pathogenicity tests with ‘Sauvignon Blanc’ detached shoots and potted vines were used to determine the virulence diversity. UP-PCR analysis determined eight genetic groups of D. mutila with 70% of the population within one group. Phylogenetic analysis also determined that New Zealand isolates were more closely related to Australian isolates than Californian isolates. Vegetative compatibility grouping analysis placed the isolates into three VCG groups, with 57% of isolates belonging to all three VCGs. Vegetative compatibility reactions were observed among isolates, but this was not correlated with the genetic clustering. Virulence assays proved that all isolates tested were pathogenic on grapevine stems. Differences in necrotic lesions lengths caused by D. mutila isolates were identified, indicating different virulence levels among isolates, however, no relationship was found between the genetic groups and the virulence. The results of the study indicated movement of D. mutila isolates between nurseries, vineyards, and other sources in New Zealand. This information will inform control strategies to limit the further spread of this pathogen into vineyards in the same region or new regions

The identity, distribution and diversity of botryosphaeriaceous species in New Zealand vineyards – a national perspective

In recent years molecular tools have been applied to provide understanding of the population structures of botryosphaeriaceous species in New Zealand vineyards. A national survey of symptomatic material from 43 vineyards showed that 88% had infection by botryosphaeriaceous species. Vine age had the strongest correlation with incidence, with the least infection in grapevines 1–5 years old (30%). Sequencing of taxonomic genes identified nine species. In contrast to other countries, N. luteum and N. parvum were predominant species with Lasiodiplodia theobromae notably absent. As with other countries, research showed that distribution is likely to be related to climate. Analysis of populations demonstrated that, despite predominantly asexual reproduction, the genetic diversity of isolates within species was high. Frequent hyphal anastomoses and fusions were observed in dual culture with weak vegetative compatibility barriers. This indicated the likelihood of frequent parasexual recombination. The isolation of genetically similar isolates from single lesions reinforced this hypothesis. A suite of molecular tools were developed to aid epidemiology studies. Endogenous markers produced for isolates with typical pathogenesis showed they could be dispersed at least 2 m from the site of conidiation in a single rain/wind event. The use of a multi-genus PCR-SCP system showed that N. parvum and N. luteum are released year round and this probably contributes to their successful invasion of vineyards. Application of these molecular tools has provided a comprehensive snapshot of New Zealand vineyards revealing a thriving and diverse population of botryosphaeriaceous species that present a serious concern to the industry

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)

The Botryosphaeriaceae: genera and species known from culture

In this paper we give an account of the genera and species in the Botryosphaeriaceae. We consider morphological characters alone as inadequate to define genera or identify species, given the confusion it has repeatedly introduced in the past, their variation during development, and inevitable overlap as representation grows. Thus it seems likely that all of the older taxa linked to the Botryosphaeriaceae, and for which cultures or DNA sequence data are not available, cannot be linked to the species in this family that are known from culture. Such older taxa will have to be disregarded for future use unless they are epitypified. We therefore focus this paper on the 17 genera that can now be recognised phylogenetically, which concentrates on the species that are presently known from culture. Included is a historical overview of the family, the morphological features that define the genera and species and detailed descriptions of the 17 genera and 110 species. Keys to the genera and species are also provided. Phylogenetic relationships of the genera are given in a multi-locus tree based on combined SSU, ITS, LSU, EF1-α and β-tubulin sequences. The morphological descriptions are supplemented by phylogenetic trees (ITS alone or ITS + EF1-α) for the species in each genus.We would like to thank the curators of the numerous fungaria and Biological Resource Centres cited in this paper, for making specimens and cultures available for examination over the past 15 yr, without which this study would not have been possible. Part of this work was supported by Fundação para a Ciência e a Tecnologia (Portugal) through grant PEst-OE/BIA/UI0457/2011. Artur Alves and Alan Phillips were supported by the programme Ciência 2008, co-funded by the Human Potential Operational Programme (National Strategic Reference Framework 2007–2013) and the European Social Fund (EU).publishe

Real-Time PCR Assays for the Detection of Puccinia psidii

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules

Co-infection by Neofusicoccum luteum and N.parvum influences direction of lesion expansion but not total lesion size

In a national survey of symptomatic vines a total of 238 samples were collected yielding 336 isolates of botry-osphaeriaceous species. From this collection 18 lesions were systematically sampled to determine if multiple species were present. Twelve lesions contained multi-ple species. Diplodia seriata was found most frequently in combination with other species (40%) but Neofusico-cum luteum and N. parvum were found most frequently together (20%). To determine whether co-infection by these two Neofusicoccum species was synergistic, trunks of 1-year-old Sauvignon blanc vines were co-inoculated with two isolates each of N. luteum and N. parvum, either together or alone. Each pair of isolates for each species consisted of a weakly virulent and highly virulent isolate, with virulence established in previous experiments. Following inoculation the presence of both species in one lesion was confirmed by PCR. The results showed that in co-inoculated lesions mean total lengths were not larger than the lesion length produced by the most virulent of the isolate pair (P≤0.05), irrespective of the virulence of the second isolate. However, when the distances from the inoculation points to lesion edges were analysed, results showed that there were significantly greater lengths below than above inoculation points for all combinations (P≤0.05). In addition, co-inoculation of two weakly virulent isolates reduced upward movement (P≤0.05). A decrease in the endophytic movement of N. luteum beyond the lesion was also observed in all co-inoculations. Overall, the results demonstrated that there were no synergistic effects of co-inoculation with these two common species

Developing molecular markers for grapevine trunk diseases

This poster depicts research focussed on developing and optimising molecular markers as tools to underpin applied research on pathogens in the viticulture industry. This study’s results for Phaeomoniella chlamydospora (Petri disease), Cylindrocarpon sp. (Black foot) and Botryosphaeria spp. (Trunk dieback and decline) are given

Genetic analysis of Neofusicoccum parvum and N. luteum isolates from nurseries and vineyards indicates different infection sources

Surveys in 2007–08 showed that Neofusicoccum parvum and N. luteum are the two most prevalent and virulent species found in New Zealand vineyards and nurseries. However, N.parvum is more common in vineyards while N. luteum dominates in nurseries. Pathogenicity studies also showed that N. parvum was more aggresive and produced more severe, darker external lesions on canes than N. luteum. This study used genetic data to elucidate population origins of N. parvum and N. luteum from vineyards and nurseries. Vineyard and nursery isolates of N. parvum (n=79) and N. luteum (n=64) were genotyped using five universally-primed polymerase chain reaction (UP-PCR) primers. The five primers were able to amplify a total of 51 loci for N. parvum (66% polymorphic) and 54 loci for N. luteum (44% polymorphic). Phylogenetic analysis using parsimony (PAUP) showed that 92% and 78% of the N. parvum and N. luteum populations, respectively, were of unique genotypes. The neighbour joining trees showed that N. parvum from nurseries clustered separately from the vineyard isolates indicating the two populations were genetically distinct. This supported the initial hypothesis that the nursery infections by this aggressive species were intercepted during the grading process and, therefore, there was less movement of this species to the vineyards. In contrast, the N. luteum nursery and vineyard populations showed high genetic similarities. This indicated that the less distinct symptoms caused by this species are not graded out and, therefore, these infections can easily migrate from the nurseries to the vineyard

Using bacterial endophytes from a New Zealand native medicinal plant for control of grapevine trunk diseases

Botryosphaeriaceous species are the causal agent of grapevine (Vitis vinifera) trunk diseases with very few options available for their control. They are also common endophytes in mānuka (Leptospermum scoparium), a New Zealand native medicinal plant, but there is no evidence of pathogenicity in this host. International research has demonstrated that endophytic bacteria can produce antimicrobial metabolites in planta. Thus, endophytic bacteria from mānuka may be viable options for biocontrol of botryosphaeriaceous species. This study was aimed at elucidating whether endophytic bacteria from mānuka with biocontrol activities can be transferred to grapevine as a heterologous host and express their bioactivity. Ten endophytic bacteria from a collection of 330 bacteria produced diffusible and volatile compound(s) that inhibited growth of six botryosphaeriaceous species. A combination of spontaneous rifampicin mutants and ERIC PCR was used to confirm endophytic colonization of the introduced antagonistic bacteria in planta. The results showed that two isolates, Pseudomonas sp. I2R21 and Pseudomonas sp. W1R33, were transferable from mānuka to grapevine when inoculated onto wounds, where they inhibited colonization by two botryosphaeriaceous species, Neofusicoccum luteum and N. parvum, respectively. The endophytic bacteria reduced lesion length caused by the pathogens (32–52%) compared to untreated controls. This study indicated that mānuka can provide a new source of microorganisms for use in sustainable agriculture