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. 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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. 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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

Botryosphaeriaceous infection in New Zealand vineyards: Identification, population structure and genetic diversity

A survey of 43 vineyards from six wine growing regions in New Zealand collected 238 grapevine wood samples displaying characteristic symptoms, including cankers, trunk necrosis, dieback and decline. Isolation from the symptomatic material showed that botryosphaeriaceous species infection was present in 88% of the vineyards and 68% of the 238 samples, which yielded 336 isolates of botryosphaeriaceous species. The incidence of infection varied between regions (P<0.001), being highest in Gisborne (96%) and lowest in Otago (23%), and between age groups (P<0.001), being highest in grapevines 6-10 years old. Infection incidence differed between scion varieties (P<0.001), being highest in Sauvignon blanc and higher in grafted than non-grafted grapevines (P<0.001). Analysis of all isolates using morphological characteristics and the molecular tool ARDRA identified nine species as Neofusicoccum parvum, N. luteum, N. australe, N. ribis, Diplodia mutila, D. seriata, Dothiorella sarmentorum, Botryosphaeria dothidea and also Do. iberica, which was a first record in New Zealand. The relative frequencies of these species showed that N. parvum was predominant (34%) followed by D. mutila (18%), D. seriata (16%), N. luteum (14%), N. australe (11%), N. ribis (3%), Do. iberica (2%), Do. sarmentorum (1%) and B. dothidea (1%). Neofusicoccum parvum and N. luteum were isolated most frequently from the North Island, and N. australe most frequently from the South Island. The Diplodia species were mostly found in South Island vineyards. The species, N. parvum, N. luteum, N. australe, D. mutila and D. seriata, varied significantly (P<0.05) in their optimum temperatures for maximum growth rate. High genetic diversity intra- and inter-vineyard and between regions was demonstrated for populations of N. parvum, N. luteum, N. australe and D. mutila using UP-PCR and neighbour joining analysis. Nei’s genetic diversity using data from 8 UP-PCR primers for N. parvum was H=0.2581 and with 5 UP-PCR primers for N. luteum, N. australe and D. mutila populations was 0.1791, 0.2417 and 0.2347, respectively. Vegetative compatibility tests with 11 N. parvum isolates selected from different branches of the neighbour joining tree showed incompatible, partially compatible and compatible interactions, with overlap between three of the four VCGs identified. For D. mutila the 14 isolates within three VCGs largely overlapped between groups. Microscopic analysis of different compatibility reactions for N. parvum and D. mutila revealed many anastomoses within and between the isolates’ colonies. Pathogenicity varied between isolates of N. parvum, N. luteum, N. australe and D. mutila in assays with excised grapevine green shoots and 1 year old potted grapevines. However, the isolates were ranked in a similar order in both assays with respect to their mean lesion lengths. On potted grapevines, the greatest ranges in lesion lengths were caused by isolates of N. parvum (0-56 mm) and N. australe (47-139 mm). On potted vines, N. luteum isolates caused the largest lesions (100-178 mm) with the greatest endophytic movement (153-268 mm) beyond lesions. Isolates from the same genetic groups in N. parvum and N. luteum neighbour joining tree generally showed similar pathogenicity levels. Co-inoculation with N. parvum and N. luteum isolates on potted grapevine showed that the infections were not synergistic or competitive, although co-inoculation caused greater downward movement from the inoculation point than single inoculations. Preliminary assays showed that N. parvum isolates could produce laccase in vitro but the variable levels from different isolates showed no relationship with their pathogenicity. From the UP-PCR fingerprints, endogenous markers were identified in N. parvum isolate B2141 and N. luteum isolate G51a2, so PCR assays were developed to identify them in environmental samples. For N. parvum B2141, a nested PCR-RFLP based on sequence polymorphism allowed the TaqI restriction endonuclease to differentiate it from other N. parvum isolates and to detect as little as 0.5 pg genomic DNA. For N. luteum G51a2, a standard PCR assay at an annealing temperature of 63ºC amplified a unique 510 bp product for isolate G51a2 but not for other N. luteum isolates, and had a sensitivity of 5 pg of genomic DNA. Neither assay amplified bands in the other botryosphaeriaceous species tested. In a field experiment with conidia of these endogenous markers of N. parvum and N. luteum, the method could detect rainwater splashed propagules up to 2 m from the conidium source

Development of isolate-specific markers for Neofusicoccum parvum and N. luteum and their use to study rainwater splash dispersal in the vineyard

Unique bands were identified in single isolates of Neofusicoccum parvum and Neofusicoccum luteum using universally primed polymerase chain reaction (UP-PCR) analysis of isolates obtained from grapevines and non-grapevine hosts in New Zealand, Australia, South Africa and the USA. Primers were designed to amplify a 1550 bp portion of the 1573 bp marker band from N. parvum isolate B2141 and a 510 bp portion of the 524 bp marker band from N. luteum isolate G51a2. A PCR-RFLP assay was developed to distinguish the N. parvum isolate B2141 from other N. parvum isolates, based on a polymorphism found in the marker band using the TaqI restriction endonuclease. For N. luteum isolate G51a2, the designed primers were specific at an annealing temperature of 63°C in the PCR. The sensitivity threshold of the N. parvum and N. luteum isolate-specific markers was 50 pg and 5 pg, respectively, when used in standard PCR with purified genomic DNA. The sensitivity of the N. parvum isolate-specific marker was increased to 0·5 pg by nested PCR. The specificity test of both isolate-specific markers with six other Botryosphaeriaceae spp. showed that they were specific to their respective species and isolates. Both markers were able to detect the conidia of N. parvum and N. luteum marker isolates in rainwater samples collected at different distances from an inoculation point in the vineyard. The results showed that rain splash could disperse the conidia of both of these species up to 2 m from the inoculum point in a single rainfall event

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