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Trichoderma asperellum Samuels, Lieckf. & Nirenberg is one of several Trichoderma species comprising effective biocontrol strains. One of these, Trichoderma asperellum T34, shows activity against soilborne pathogens such as Rhizoctonia, Fusarium, and Pythium. The activity of biocontrol strains such as T34 depends on their establishment in the rhizosphere. Monitoring of the population of T34 and other strains in the rhizosphere and the growing medium is therefore important for evaluating their potential in different crops and different growing systems. Until now, strain-specific monitoring has been very difficult as the main quantification methods, i.e. traditional dilution plating or standard molecular detection methods, are specific only at genus or species level, respectively. Genotyping-by-sequencing was used to identify genomic loci specific to T34. These were used to develop strain-specific Taqman real-time (qPCR) assays. After testing for specificity and sensitivity, two assays were combined in duplex format. The duplicate assay provided an internal control to avoid a false positive signal in case one of the target loci would be present in an as yet unidentified non-target organism. An optional enrichment step in semi-selective medium was included for increased sensitivity in cases of small T34 population size, theoretically allowing detection down to 0.1 spore equivalent per mL growing medium. The techniques were applied to 200 mL samples of growing medium analyzed 0, 19, and 112 days after spiking with different numbers and combinations of spores from target and non-target Trichoderma isolates. This technique shows potential for developing other strain-specific detection assays using an affordable genotyping tool. [ABSTRACT FROM AUTHOR]

Phytophthora pseudosyringae is a self‐fertile pathogen of woody plants, particularly associated with tree species from the genera Fagus, Notholithocarpus, Nothofagus and Quercus, which is found across Europe and in parts of North America and Chile. It can behave as a soil pathogen infecting roots and the stem collar region, as well as an aerial pathogen infecting leaves, twigs and stem barks, causing particular damage in the United Kingdom and western North America. The population structure, migration and potential outcrossing of a worldwide collection of isolates were investigated using genotyping‐by‐sequencing. Coalescent‐based migration analysis revealed that the North American population originated from Europe. Historical gene flow has occurred between the continents in both directions to some extent, yet contemporary migration is overwhelmingly from Europe to North America. Two broad population clusters dominate the global population of the pathogen, with a subgroup derived from one of the main clusters found only in western North America. Index of association and network analyses indicate an influential level of outcrossing has occurred in this preferentially inbreeding, homothallic oomycete. Outcrossing between the two main population clusters has created distinct subgroups of admixed individuals that are, however, less common than the main population clusters. Differences in life history traits between the two main population clusters should be further investigated together with virulence and host range tests to evaluate the risk each population poses to natural environments worldwide. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum f.sp. lactucae (Fol) causes a vascular disease in lettuce that results in significant yield losses. Race-specific and sensitive real-time PCR assays were developed for Fol races 1 and 4, which are prevalent in Europe. Using genotyping-by-sequencing, unique DNA loci specific to each race were identified and subsequently used for the design of primers and hydrolysis probes. Two assays per race were developed to ensure specificity. The two assays of each race could be run in duplex format, while still giving a sensitivity of 100 fg genomic DNA for all assays. Sample preparation methods were developed for plant tissue, soil, and surfaces, with an extra enrichment step when additional sensitivity was required. By controlling the incubation conditions during the enrichment step, the real-time PCR signal could be matched to the number of spore equivalents in the original sample. When enriching naturally infested soil, down to six conidiospore equivalents L-1 soil could be detected. As enrichment ensures sensitive detection and focuses on living Fol propagules, it facilitates the evaluation of control measures. The developed detection methods for soil and surfaces were applied to samples from commercial lettuce farms and confirmed the prevalence of Fol race 4 in Belgium. Monitoring of soil disinfestation events revealed that despite a dramatic decrease in quantity, the pathogen could still be detected either immediately after sheet steaming or after harvesting the first new crop. The detection method for plant tissue was successfully used to quantify Fol in lettuce inoculated with race 1, race 4 or a combination of both. Under the temperature conditions used, race 4 was more aggressive than race 1, as reflected in larger amounts of DNA of race 4 detected in the roots. These newly developed assays are a promising tool for epidemiological research as well as for the evaluation of control measures. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum f. sp. lactucae race 4 causes vascular necrosis and wilting of lettuce. First observed in Belgium in 2015, the lack of disease resistance in commercial cultivars allowed this pathogen to spread to nearly the entire Belgian production area within 4 years. Different levels of disease development were observed in different commercial greenhouses. To help explain this variation, we collected 78 Fusarium isolates and characterized them both physiologically and genetically. Molecular race identification indicated that 91% of the isolates belonged to race 4, while 6% of the isolates belonged to race 1, which was not previously reported in Belgium. Pathogenicity assays using differential cultivars confirmed the molecular race assignment of selected isolates. Cultivar Patriot was identified as a suitable new differential cultivar to race 4. Race 4 isolates were more aggressive than race 1 isolates at 24°C, but only when using chlamydospore inoculum instead of a root dip assay containing microconidia. Variation in pathogenicity and aggressiveness of the races may explain differences in disease development in commercial greenhouses. Based on genotyping‐by‐sequencing (GBS), race 1 and race 4 isolates were highly similar to reference isolates. Fusarium curvatum, F. oxysporum f. sp. tulipae and F. oxysporum f. sp. rhois were phylogenetically separated from F. oxysporum f. sp. lactucae races 1 and 4 based on the GBS data, but not when using multilocus sequence data. Within F. oxysporum f. sp. lactucae race 4, the GBS data differentiated two rather homogeneous groups, suggesting at least two introductions. However, the two groups did not differ in aggressiveness. [ABSTRACT FROM AUTHOR]

Invasive, exotic plant pathogens pose a major threat to native and agricultural ecosystems. Phytophthora × cambivora is an invasive, destructive pathogen of forest and fruit trees causing severe damage worldwide to chestnuts (Castanea), apricots, peaches, plums, almonds and cherries (Prunus), apples (Malus), oaks (Quercus), and beech (Fagus). It was one of the first damaging invasive Phytophthora species to be introduced to Europe and North America, although its origin is unknown. We determined its population genetic history in Europe, North and South America, Australia and East Asia (mainly Japan) using genotyping-by-sequencing. Populations in Europe and Australia appear clonal, those in North America are highly clonal yet show some degree of sexual reproduction, and those in East Asia are partially sexual. Two clonal lineages, each of opposite mating type, and a hybrid lineage derived from these two lineages, dominated the populations in Europe and were predominantly found on fagaceous forest hosts (Castanea, Quercus, Fagus). Isolates from fruit trees (Prunus and Malus) belonged to a separate lineage found in Australia, North America, Europe and East Asia, indicating the disease on fruit trees could be caused by a distinct lineage of P. × cambivora, which may potentially be a separate sister species and has likely been moved with live plants. The highest genetic diversity was found in Japan, suggesting that East Asia is the centre of origin of the pathogen. Further surveys in unsampled, temperate regions of East Asia are needed to more precisely identify the location and range of the centre of diversity. [ABSTRACT FROM AUTHOR]

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters—diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility—were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes. [ABSTRACT FROM AUTHOR]

Site-specific high throughput monitoring of airborne ascospores of Venturia inaequalis, the causal agent of apple scab, can improve existing warning systems. A new qPCR assay was developed to quantify ascospores collected by a simple rotating-arm spore sampler. The qPCR assay was highly specific and sensitive, with a limit of quantification of 20 ascospores per sample. The new detection system was compared to sampling with a traditional Burkard volumetric spore trap and to microscopic quantification. During controlled ascospore release experiments in a closed environment, strong correlations (ρ: 0.96 to 0.99) were observed between the two types of samplers and the two methods of quantification but significantly larger numbers of spores (log difference: 0.43 to 0.69) were obtained when using the rotating-arm sampler and when using molecular quantification. During comparisons under outdoor conditions over a three-year period, reasonable correlations between the techniques (average ρ = 0.61) were observed. When rotating-arm samplers operate continuously they can get saturated but their counts still correlated better with those from the Burkard sampler than when they only operate during rain and until two hours after. This suggests that ascospores were also captured outside of rain events. Based on these comparisons, molecular quantification of spores captured with the rotating-arm sampler appears to be a sensitive and reliable method to determine airborne ascospores of V. inaequalis and holds promise as a tool to guide targeted fungicide applications in commercial orchards as well as to increase our knowledge of the aerobiology of this pathogen. [ABSTRACT FROM AUTHOR]

Box blight is a widespread disease of Buxus caused by the pathogen Calonectria pseudonaviculata. It is responsible for significant losses in nurseries, gardens and wild boxwood populations. Our goal was to maximize the efficiency of a breeding programme towards increased disease resistance. The use of artificial inoculation of young F1 seedlings with C. pseudonaviculata spores under greenhouse conditions appeared to be a reliable tool for early selection of interesting prebreeding material. Overall, the four hybrid populations screened showed a segregating behaviour between their parents when determining the percentage of diseased leaves and lesion diameter. Genotypes were also found with an increased tolerance as compared to the parental species. Approximately 50% of the seedlings had the same score for both parameters after artificial inoculation in the greenhouse and in the field. Of the seedlings that showed severe symptoms in the greenhouse, <15% showed no disease symptoms in the field. Therefore, for larger breeding programmes, we propose a two‐step selection procedure: first artificial inoculation at seedling level to eliminate all genotypes with severe symptoms and then evaluation of the remaining seedlings in the field. Using this strategy, we were able to select several genotypes in our four hybrid populations with improved resistance to C. pseudonaviculata. [ABSTRACT FROM AUTHOR]

An international test performance study (TPS) was organised to generate validation data for three molecular Synchytrium endobioticum tests: van den Boogert et al. (European Journal of Plant Pathology 113, 47-57, 2005), and van Gent-Pelzer et al. (European Journal of Plant Pathology, 126, 129-133, 2010) for the detection of S. endobioticum, and the pathotype 1(D1) identification test described by Bonants et al. (European Journal of Plant Pathology, 143, 495-506, 2015). Two TPS rounds were organised focussing on different test matrices, i.e. round 1: warted potato tissue, and round 2: resting spore suspensions. When using the tests for detection and identification of S. endobioticum in warted potato tissue, no significant differences were observed for diagnostic sensitivity, diagnostic specificity, overall accuracy, analytical sensitivity and robustness. When using the tests for detection and identification of S. endobioticum in resting spore suspensions, the van den Boogert and van Gent-Pelzer tests significantly outperform the Bonants test for diagnostic sensitivity and diagnostic specificity. For overall accuracy and analytical sensitivity, the van Gent-Pelzer significantly outperforms the van den Boogert and Bonants tests and is regarded as the test of choice when identifying S. endobioticum from resting spores. Tests regarded fit for purpose for routine testing of wart material and resting spore suspensions are proposed for the update of EPPO standard PM7/28(1) Synchytrium endobioticum. [ABSTRACT FROM AUTHOR]

All Phytophthora ramorum EU1 lineage isolates tested are of A1 mating type, except for three rare isolates from 2002 to 2003 from Belgium, which were originally assigned the A2 mating type. In one of these isolates (2338), a switch from A2 to A1 mating type was observed in 2006. This observation initiated a larger study in which all cultures and subcultures of the original three EU1 A2 isolates, maintained in three laboratories under different storage conditions, were checked for mating type change. The A2 to A1 mating type switch was observed in four of seven independently maintained isolates that were derived from isolate 2338 in two laboratories, using different transfer regimes and storage conditions. Following the mating type switch to A1 in these four derived isolates, no reversion back to A2 mating was observed, even after up to 5 years of additional isolate maintenance and several more subculturing events. The three other isolates that were derived from isolate 2338 as well as the other EU1 A2 isolates collected in 2002 and 2003 and stored in the same conditions did not display such mating type change. The potential causes of the mating type conversions as well as their epidemiological implications are discussed. [ABSTRACT FROM AUTHOR]

Fusarium foetens is a recently described aggressive vascular pathogen of Begonia x hiemalis. Since 2004, it has caused severe losses for Begonia growers in Northern Europe and North America. F. foetens is likely to be of exotic origin. Little is known about the accumulation of the fungus in Begonia plants before and during symptom expression and about its host range. We have optimised a molecular detection method for F. foetens by only using the plant part containing the largest amount of the pathogen and by optimising the tissue maceration and DNA extraction techniques. This allowed a reliable detection limit of 2310 spore equivalents per plant and a theoretical detection limit of as low as 84 to 167 spore equivalents per plant. Using this method, we demonstrated exponential accumulation of F. foetens DNA in Begonia roots, resulting in symptoms at a threshold of approximately 10 spore equivalents and levelling off at 10 spore equivalents per plant. The observed rate of accumulation and the amount of pathogen DNA in non-symptomatic plants can be combined to determine whether the cuttings were infected after delivery at the Begonia nursery and to calculate the estimated timing of symptom development. To test the host range, we applied the optimised molecular detection technique. During these tests, only Begonia x hiemalis plants became symptomatic, but many other plant species supported growth of the pathogen. This information can be used to aid pathogen control and has implications for pest risk assessment. [ABSTRACT FROM AUTHOR]

Phytophthora ramorum is a recently introduced pathogen in Europe and North America consisting of three clonal lineages. Due to the limited intralineage genetic variation, only a few polymorphic markers are available for use in studies involving the epidemiology and evolution of P. ramorum. A total of 159 primer pairs for candidate polymorphic SSR loci were tested with universal labeling. Four polymorphic microsatellite loci were identified within the NA1 lineage and one within the NA2 lineage, demonstrating the power and flexibility of the screening technique. The markers may significantly increase the number of genotypes that can be identified and as such can help better characterize the North American lineages of P. ramorum. [ABSTRACT FROM AUTHOR]

Puccinia horiana is the causal agent of chrysanthemum white rust or Japanese rust. This microcyclic autoecious rust has a quarantine status and can cause major damage in the commercial production of Chrysanthemum x morifolium. Given the international and often trans-continental production of planting material and cut flowers of chrysanthemum and the decreasing availability of registered fungicides in specific regions, breeding for resistance against P. horiana will gain importance and will need to involve the appropriate resistance genes for the pathotypes that may be present. As pathotypes have not been well characterized in this system, the main objective was to build an international collection of isolates and screen these on a large collection of cultivars to identify different pathotypes. Using a robust and high throughput bioassay, we tested 36 selected cultivars with 22 individual single-pustule isolates of P. horiana. The isolates originated from three different continents over 4 different collection years and included some isolates from cultivars previously reported as resistant. In most cases the bioassays resulted in a clear scoring of interaction phenotypes as susceptible or resistant, while in several cases consistent intermediate phenotypes were found, often on specific cultivars. Twenty-four of the cultivars gave a differential interaction phenotype profile. All isolates produced a unique profile, infecting a minimum of 4 and a maximum of 19 differential cultivars. Based on the Person analysis of these profiles, this pathosystem contains at least seven resistance genes (and seven avirulence genes), demonstrating the highly complex race structure in this pathosystem. [ABSTRACT FROM AUTHOR]

Phytophthora ramorum, a recently described North American and European pathogen, has three clonal lineages. The NA1 and NA2 lineages are found in North American forests and nurseries, while the EU1 lineage appears mainly in European nurseries. P. ramorum is heterothallic, having two mating types A1 and A2. All NA1 and NA2 isolates are of A2 mating type. When first collected, all EU1 isolates were of A1 mating type, with the exception of one A2 isolate collected in Belgium in 2002. Screening 410 other Belgian isolates for mating type revealed two additional EU1-A2 isolates collected in 2002 and 2003. PCR-RFLP, AFLP and SSR markers were used to determine the nature of the mating type change. The three isolates show no indications of sexual recombination or mitotic crossing over, indicating that mutation or mitotic gene conversion is the most likely explanation for the mating type change. We compared the pathogenicity and sporulation characteristics of the EU1-A2 isolates to those of EU1-A1 and NA1-A2 isolates on four host plants. Despite small differences in pathogenicity on some hosts, the EU1-A2 isolates were similarly aggressive to each other and to the EU1-A1 isolates and more aggressive than the NA1-A2 isolates. Sporulation characteristics were also comparable among EU1-A2 isolates and between EU1-A1 and EU1-A2 isolates, except for EU1-A2 isolate BBA 26/02. The limited genotypic and phenotypic differences between EU1-A2 isolates probably evolved after the mating type change, which may have occurred several years before the isolates were detected. There are strong indications that the EU1-A2 population has been eradicated from Belgium. [ABSTRACT FROM AUTHOR]

Summary One stage in the symbiotic interaction between the bacterium Xenorhabdus nematophila and its nematode host, Steinernema carpocapsae , involves the species-specific colonization of the nematode intestinal vesicle by the bacterium. To characterize the bacterial molecular determinants that are essential for vesicle colonization, we adapted and applied a signature-tagged mutagenesis (STM) screen to this system. We identified 15 out of 3000 transposon mutants of X. nematophila with at least a 15-fold reduction in average vesicle colonization. These 15 mutants harbour disruptions in nine separate loci. Three of these loci have predicted open reading frames (ORFs) with similarity to genes ( rpoS , rpoE , lrp ) encoding regulatory proteins; two have predicted ORFs with similarity to genes ( aroA , serC ) encoding amino acid biosynthetic enzymes; one, designated nilB ( n ematode i ntestine l ocalization), has an ORF with similarity to a gene encoding a putative outer membrane protein (OmpU) in Neisseria ; and three, nilA , nilC and nilD , have no apparent homologues in the public database. nilA , nilB and nilC are linked on a single 4 kb locus. nilB and nilC are > 10 4 -fold reduced in their ability to colonize the nematode vesicle and are predicted to encode membrane-localized proteins. The nilD locus contains an extensive repeat region and several small putative ORFs. Other than reduced colonization, the nilB , nilC and nilD mutants did not display alterations in any other phenotype tested, suggesting a specific role for these genes in allowing X. nematophila to associate with the nematode host. [ABSTRACT FROM AUTHOR]

The article focuses on a proposal to change the name of pathogen causing box blight Cylindrocladium buxicola by its earlier name Cylindrocladium pseudinaviculatum due to unavailability of any evidence regarding the species Calonectria. It informs that defoliation caused by these species can kill the plants especially young seedlings and was first detected in the year 1994 in England.