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Phoma stem canker / blackleg is an internationally important disease of Brassicas including B. napus (oilseed rape, OSR), caused by multiple genetic subclades of the fungi Plenodomus lingam (formerly Leptosphaeria maculans) and P. biglobosus (L. biglobosa). In Spring 2021, Phoma-like disease symptoms were observed on leaves and stems of Eutrema japonicum (wasabi) crops at three UK sites (Northern Ireland, Southern England and the West Midlands). Fungal isolation from wasabi leaf spots yielded colonies with two distinct phenotypes on potato dextrose agar (PDA). Isolates from the Northern Ireland and Southern England sites had white colonies with abundant pink cirri that were confirmed (based on ITS rDNA, beta tubulin and actin sequences) as P. biglobosus subclade ‘canadensis’ (Pbc). Those from the West Midlands site, however, had yellow pigmented colonies and were confirmed by sequencing as P. biglobosus subclade ‘brassicae’ (Pbb). Greenhouse pathogenicity testing showed that Pbb and Pbc wasabi isolates were pathogenic not only to this host but also OSR, B. oleracea (cabbage), and B. rapa (pak choi). Re-isolation of the fungi was attempted and confirmed from lesions that developed on inoculated OSR and wasabi, thus completing Koch’s postulates. These findings represent new discoveries for both Pbb and Pbc on wasabi, plus for Pbc in Europe. The crop health implications of these results are briefly considered.

International trade in live plant materials has increased worldwide over the last four decades. This has led to a dramatic increase in the introduction, establishment and spread of non-native plant pathogens. Regulatory authorities need advice on measures that may mitigate these adverse consequences of trade. Risk models may be used to underpin such advice. In this review, we give a systematic overview of the data needed for a quantitative risk model for Phomopsis vaccinii, which causes stem and fruit infections on Vaccinium species, and sometimes death, potentially also on native wild Vaccinium species in the EU. P. vaccinii is a quarantine organism worldwide, except for North America, where it is endemic. Despite extensive knowledge of the aetiology of the diseases caused by this pathogen and its taxonomy, quantitative data on transportation and detection of infected plants for planting and berries are scarce, and quantitative assessment of the future introduction, establishment and spread of P. vaccinii is difficult. Estimation of the potential impact of this pathogen in production fields and wild Vaccinium stands is even more difficult. P. vaccinii is not unique in this respect, and this review indicates the need for more and better quantitative data for assessment of the risks posed by newly introduced plant pathogens in areas where they are not endemic.

The project was a fusion of engineering, IT, electronics, molecular biology, aerobiology and field biology, which aimed to produce an automated air sampling device, able to use a DNA-based method to detect spores of key pathogens or genetic traits such as fungicide insensitivity, and text the result, along with met data, to a web-based information portal to alert growers and advisers. The system was designed for multiple devices to operate as a network to enhance information quality and IT systems were also designed to augment currently available infection models with the information on airborne spore presence to produce a disease risk alert in time for application of crop protection products. The devices are powered by mains electricity so each one can be placed in a secure location on a farm, close to crops, ready to detect and report the first signs of spore release long before crop infection. This was found to be preferred by consulted end-users over placing a battery-powered device in a remote field site, due to security concerns and previous data from high volume samplers suggests that airborne inoculum affecting broad-acre crops in the proximity of the farm scale can be detected easily in this way (West et al, 2017). The mains-powered automated air sampler was designed and built by the Burkard Manufacturing Co. Ltd., comprising a high-volume air sampler, which samples air at 300L/minute and can collect spores at least as small as 4 µm (aerodynamic diameter) with excellent efficiency (>90%). Each device is programmed by the user to sample for a set period each day. The collected sample is then processed mechanically in liquid to burst all spore types, releasing DNA, and a sub-sample of the disrupted spore suspension is transferred to a reaction tube where an isothermal DNA-based assay takes place inside the sampler. Two types of DNA-based assay can be used, LAMP assays or RPA assays, which differ in the reagents used and the temperature at which the reaction takes place, around 65-72 or 37-40 ֯C, respectively. The result of the assay is measured by fluorescence and results are sent wirelessly by an internal 4G router as a text message to a server. This can be repeated, currently for up to three different target pathogens to be tested from the same sample each day. Weather data, collected continuously by an on-board met station, is also sent by text every 10 minutes. The resulting data is automatically processed, collated and graphed for display on a web-portal. Simple rules applied to the data allow an automated calculation of the spore detection result, indicating zero, low or high numbers of spores present and an estimation of occurrence of infection conditions can currently be made for yellow rust and Sclerotinia. Hence, the system can provide information in near real-time on the presence of airborne spores and the weather conditions necessary for infection. Each device requires weekly attendance by the user to replenish consumables (reagents and various tubes) and to perform simple maintenance. In future, additional new assays will be added and additional weather-based infection models will be integrated. The project suffered a major and unforeseen technical problem concerning translating effective lab-based assays using freshly prepared wet reagents into stable, dried formats with long shelf-life and good reliability that could be used in the autoDNA-sampler. Solutions to the problem involved different drying processes, addition of stabilising chemicals and testing of different reagents. A completely dry format was successfully developed for one assay as part of a related project but unfortunately this did not work reliably for arable crop pathogen assays when validated against freshly-made wet reagents in a lab. Instead, the best working development by the end of the project was a compromise method involving some dried reagents and some separately-stored liquid reagents that have a moderate shelf life (several weeks at room temperature) and can be added successfully to additional dried reagents along with the spore sample with good sensitivity and specificity of results. Despite the technical problem, this ambitious project has developed new LAMP assays for lab-based use to detect the following pathogens: Pyrenopeziza brassicae (King et al, 2017), and publications are in preparation for the following: Sclerotinia sclerotiorum, Zymoseptoria tritici, Rhynchosporium sp, Oculimacula yallundae & O. acuformis (joint assay), Alternaria solani, and for fungicide insensitivity in Zymoseptoria tritici (assays for reduced DMI sensitivity and separately an assay for multidrug resistance including reduced DMI and SDHI sensitivity). These new assays are themselves of great use for research purposes and also for practical use by extension workers and growers with the correct portable equipment to perform in-field diagnostic tests. In addition, some existing assays, published by other researchers, were assessed for sensitivity and specificity to UK pathogens (Phytophthora infestans, Puccinia striiformis and Fusarium graminearum). The new lab-based assays will be available for use by diagnostic providers and for research in crop protection. The automated air sampling device will continue development under other funding and is expected to be available for commercial use to improve spray timing and fungicide selection from 2019 onwards. The novel device will lead to a new approach in precision agriculture by providing information on exactly when and where growers should protect crops against disease, hence informing smart spray recommendations. The technology will in time, with use of appropriate reagents (DNA primers), be translated to improve disease control in other AHDB sectors and could be available for fungicide resistance monitoring in addition to disease forecasting. The technology, through detection of Fusarium graminearum, will assist in control of mycotoxins and will optimise agrochemical performance as part of integrated crop management. Further research is recommended to enable assessment of the system, further validate it against results from conventional air samplers for new target pathogens and to improve interpretation of results and to optimise sampler location when used for detection of specific pathogens. An additional improvement might be to add a component of forecast weather for up to three days ahead of current time whenever the device has detected airborne spores or to add economics models based on crop growth stage and value of the expected yield to assist with spray decisions.

The Panel on Plant Health performed a pest categorisation of the six-toothed bark beetle, Ips sexdentatus (B€orner) (Coleoptera: Curculionidae, Scolytinae), for the EU. I. sexdentatus is a well-defined and distinguishable species, native to Eurasia and recognised mainly as a pest of pine (Pinus spp., in the pest’s whole range) and spruce (mainly Picea orientalis in Turkey and Georgia). It also might occasionally attack Larix spp. and Abies spp. It is distributed throughout the EU (24 Member States). It is a protected zone quarantine pest in Ireland, Cyprus and the United Kingdom (Northern Ireland, Isle of Man), listed in Annex IIB of Council Directive 2000/29/EC. Wood, wood products, bark and wood packaging material are considered as pathways for this pest, which is also able to disperse by flight over tens of kilometres. The adults normally establish on fallen or weakened trees (e.g. after a fire or a drought) and can also massattack healthy trees. The males produce aggregation pheromones that attract conspecifics of both sexes. The insects also inoculate pathogenic fungi to their hosts. There are one to five generations per year. The wide current geographical range of I. sexdentatus suggests that it is able to establish anywhere in the EU where its hosts are present. Sanitary thinning or clear-felling are the major control methods. Pheromone mass-trapping is also locally implemented. Quarantine measures are implemented to prevent entry into the protected zones. All criteria for consideration as potential protected zone quarantine pest are met. The criteria for considering I. sexdentatus as a potential regulated non-quarantine pest are not met since plants for planting are not viewed as a pathway.

The EFSA Panelon Plant Health performed a pest categorisation of Palm lethal yellowing phytoplasmas for the EU territory. This name is used to describe diseases that share the same succession of symptoms in palms that are caused by a number of strains of phytoplasma, for which efficient molecular detection assays are available. The pest is not known to occur in the EU and therefore does not meet one of the criteria for being a Union regulated non-quarantine pest. For Candidatus Phytoplasma palmae', the planthopper Haplaxius crudus, which is not known to be present in the EU, is the confirmed vector, but for the other strains, the vectors are unknown. The host range of the pest is restricted to Arecaceae species, in particular coconut. The pest is regulated on all known hosts in Annex IIAI of Directive 2000/29/EC. It could potentially enter the EU via plants for planting or through infected vectors. The phytoplasmas could become established in the EU as host plants are present. It is unknown whether arthropods present in the EU could be vectors. The potential impact of the pest if introduced into the EU is difficult to assess given this uncertainty but is estimated to be limited. The main knowledge gaps concern the status of potential vector insects in the EU; the possibility for seed transmission of the phytoplasmas; the origin and volume of the trade in palm seeds and plants for planting; the host status and susceptibility of many palm species grown in the EU and the potential new assignments of phytoplasmas to this categorisation that might have associated alternate hosts. Palm lethal yellowing phytoplasmas meet the criteria assessed by EFSA for consideration as Union quarantine pest. (C) 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

The EFSA Panel on Plant Health performed a pest categorisation of non‐EU isolates of Beet curly top virus (BCTV) for the European Union territory. The virus causes severe diseases in beet, tomatoes and pepper crops, occurs predominantly in warm and dry zones and is reported from many countries outside the EU in particular from western USA and Mexico. New data from complete virus genomes make BCTV a well characterised virus species of which currently 11 strains are known and for which diagnostic methods are available. BCTV has a very broad host range of more than 300 species some of which may remain symptomless. Aside from vegetative propagation of infected plants, the only mode of BCTV transmission and spread is by the leafhopper Circulifer tenellus which efficiently transmits the virus in a persistent mode and which is present in several southern EU Member States. No current reports of BCTV presence in the EU exist and because of doubts about the accuracy of older reports, BCTV likely is absent from the EU territory. BCTV can enter into the EU with viruliferous insects and with imports of plants not subject to specific EU regulation. Because both the virus and its vector have a wide host range, BCTV is expected to establish and spread in the Member States where its vector is present and to cause severe diseases in sugar beet and tomato as well as in other crops. Overall, BCTV non‐EU isolates meet all the criteria evaluated by EFSA to qualify as a Union quarantine pest and do not meet the criterion of presence in the EU to qualify as a Union regulated non‐quarantine pest (RNQP). The main uncertainties concern (1) the presence of BCTV in the EU, (2) the distribution of C. tenellus and (3) the main commodities for virus entry.

Following the 2014 EFSA’s Panel on Plant Health scientific opinion on the pest categorisation of the spider mite Eotetranychus lewisi, the European Commission requested the Panel to perform a pest risk assessment and evaluate the risk reduction options. A stochastic model was used to assess entry, establishment and spread and related uncertainties. In the EU, E. lewisi has only been reported to occur in Portugal (Madeira). Entry pathways assessed were strawberry plants for planting from the USA, poinsettia and raspberry plants for planting, and orange and lemon fruits from third countries. Entry is most likely via poinsettia. Under current EU phytosanitary requirements, there is around a one in ten chance that E. lewisi will establish outdoors over the next 10 years. Although unlikely, establishment would most likely occur in southern Europe where environmental conditions, temperature and host density, are most suitable. If E. lewisi did establish, pest spread is expected to be mainly human assisted, most likely the mite being transported long distances on plants for planting. Nevertheless, while remaining a regulated pest, spread would be slow and most likely confined to one NUTS 2 area after 10 years. Under a scenario with enhanced measures (pest free place of production) at origin, the Panel’s assessment indicate that it is extremely unlikely that E. lewisi would establish within 10 years hence spread is also extremely unlikely. The absence of trade of host plants from Madeira to other parts of the EU could explain why E. lewisi has not spread to other EU Member States. E. lewisi is reported as reducing yield and quality of peaches and poinsettia and is regarded as a growing concern for strawberry and raspberry growers in the Americas. The Panel concludes that should E. lewisi be introduced in the EU similar impacts could be expected.

As requested by the European Commission, the EFSA Panel on Plant Health (PLH) Panel assessed the risk of Diaporthe vaccinii in the EU, focusing on entry, establishment, spread and impacts on cultivated and wild Vaccinium species, the principal hosts being American and European cranberry and blueberry. Several outbreaks occurred in the EU since 1956, but most were eradicated except in Latvia. The Panel considered entry via fruits and plants for planting. The risk of establishment from discarded infected berries is much lower than from infected plants for planting, of which, potted plants and cuttings pose the greatest risk, while plug plants, derived from tissue culture and grown in pest free structures, pose a low risk. Nine per cent of the EU is highly suitable for establishment of the pathogen, mostly in the SE and NE. Following establishment, the pathogen could spread naturally over short range, and by human assistance over long range. Calculations with an integrated model for entry, establishment and spread, indicate that with current regulations, over a period of 5 years, a few hundred cultivated Vaccinium plants and several thousand Vaccinium plants in natural ecosystems would contract the disease. The associated loss of commercial production is small, less than one tonne of berries per year. On natural vegetation, the median impact after 5 years was estimated to be negligible affecting a negligible proportion of the natural Vaccinium population (2 x 10 8). However, the uncertainty of this estimate was high, due to uncertainty about the rate of spread; in a worst-case scenario (99th percentile), almost 1% of plants in natural areas would become infected. Complete deregulation (scenario A1) was predicted to increase the impact substantially, especially in natural areas, while additional measures (scenario A2) would effectively eliminate the entry of infected plants for planting, further reducing the impacts below the current situation.

Following a request from the European Commission, the EFSA Plant Health (PLH) Panel performed a pest categorisation of Gremmeniella abietina, a well-defined species and distinguishable fungus of the family Godroniaceae. The species G. abietina includes several varieties, races and biotypes that are found in different geographical locations, on different hosts and that vary in aggressiveness. The pathogen causes diseases on Pinus species and other conifers such as Abies spp., Picea spp., Larix spp. and Pseudotsuga spp. known as Scleroderris canker in North America and Brunchorstia dieback in Europe. G. abietina has been reported from 19 EU Member States, without apparent ecoclimatic factors limiting establishment. The pathogen is a protected zone (PZ) quarantine pest (Annex IIB) for Ireland and the UK (Northern Ireland). The main European hosts are widespread throughout most of the EU and have been frequently planted in the PZ. The main means of spread are wind-blown ascospores, rain-splashed conidia, plants for planting and traded Christmas trees. Given that G. abietina is most damaging to species that are grown towards the limit of their range, impacts can be expected in the PZ, should the pathogen be introduced there. Risk reduction options include selection of disease-free planting material, nursery inspections, selection of planting sites at some distance from infested plantations, appropriate spacing between plants and thinning. The main uncertainties concern the indeterminate endophytic stage of the fungus, the pathogen distribution and the future taxonomic status of G. abietina, given its intraspecific diversity. All the criteria assessed by the Panel for consideration as potential PZ quarantine pest are met. The criterion of plants for planting being the main pathway for spread for regulated non-quarantine pests is not met: plants for planting are only one of the means of spread of the pathogen.

The Panel on Plant Health performed a pest risk assessment on Radopholus similis, the burrowing nematode for the EU. The quantitative assessment focused on entry, establishment, spread and impact on tropical and subtropical ornamental host plants, the main pathways for entry of R. similis into the EU. Infested consignments are expected to enter the risk assessment area on ornamentals under all scenarios. For citrus, which is a closed pathway for entry, outdoor establishment was assessed. Establishment may only take place after successful transfer from ornamental plants to citrus production systems. This event is called ‘shift’ in this assessment, to indicate that this is an unusual transfer. It has been estimated that establishment of this nematode in the open field in the EU citrus production areas under current temperatures is possible in most parts of the citrus production area in the EU. Temperature conditions will prevent the nematode from establishing only in the northernmost citrus areas and at higher altitudes in the south. Host plants for planting originating from infested places of production (greenhouses) within the risk assessment area are considered the main pathway for spread within the risk assessment area. Under current climatic conditions, the population of R. similis is not expected to reach damaging population levels in the open field. In case of increased temperatures due to global warming, the nematode population may reach damaging levels in very few places outdoors. Currently, main impact is considered for ornamental greenhouse production in the risk assessment area. Impact will be either caused by direct plant growth reductions or loss due to phytosanitary measures applied on regulated plants. Despite the fact that R. similis is globally considered as one of the most destructive plant parasitic nematodes, the impact in the risk assessment area is considered low.

Following a request from the European Commission, the EFSA Plant Health (PLH) Panel performed a pest categorisation of Pseudocercospora pini-densiflorae, a well-defined and distinguishable fungal species of the family Mycosphaerellaceae. The regulated harmful organism is the anamorph Cercoseptoria pini-densiflorae (synonym Cercospora pini-densiflorae) with the corresponding teleomorph Mycosphaerella gibsonii. P. pini-densiflorae causes a needle blight of Pinus spp. also known as Cercospora blight of pines or Cercospora needle blight. P. pini-densiflorae is reported from sub-Saharan Africa, Central and South America, Asia and Oceania, but not from the EU. The pathogen is regulated in Council Directive 2000/29/EC (Annex IIAI) as a quarantine organism whose introduction into the EU is banned on plants (other than fruit and seeds) and wood of Pinus. The pest could enter the EU via plants for planting and other means (uncleaned seed, cut branches of pine trees, isolated bark, growing media accompanying plants, and mycorrhizal soil inocula). Hosts are widespread in the EU and favourable climatic conditions are present in Mediterranean countries. Pinus halepensis, Pinus nigra, Pinus pinea, Pinus pinaster and Pinus sylvestris are reported to be highly susceptible to the pathogen. The pest would be able to spread following establishment after introduction in the EU mainly on infected plants for planting. The pest introduction could have impacts in nurseries and young plantations. Cleaning seeds from needles and removing infected seedlings and pine litter from affected nurseries can reduce the risk of establishment in nurseries and of spread from nurseries to forests, especially given the limited scale of splash dispersal. The main knowledge gaps concern (i) the role of means of entry/spread other than plants for planting and (ii) the potential consequences in mature tree plantations and forests. The criteria assessed by the Panel for consideration as potential quarantine pest are met. For regulated nonquarantine pests, the criterion on the pest presence in the EU is not met.

Following a request from the European Commission, the EFSA Panel on Plant Health (PLH) was asked to assess two publications, authored by Magarey et al. and Martinez-Minaya et al. from 2015, with regard to a need to update the EFSA Scientific Opinion from 2014 on the risk of Phyllosticta citricarpa (Guignardia citricarpa) for the EU territory. The EFSA PLH Panel was also requested to assess any other relevant scientific information published after the finalisation of the EFSA Scientific Opinion. The fungus P. citricarpa (McAlpine) Van der Aa causes the citrus disease citrus black spot (CBS), and is regulated as quarantine organism in Council Directive 2000/29/EC. The Panel assessed the two publications in detail as well as all relevant publications published until 31 March 2016. A comparison with the EFSA PLH Panel (2014) was made, survey data on CBS from South Africa used in Magarey et al. (2015) were evaluated, and the citrus production areas in the EU were characterised and compared with results from Magarey et al. (2015). Uncertainty and model sensitivity were discussed. It was concluded that the evidence presented in Magarey et al. from 2015 does not require an updating of EFSA PLH Panel (2014). The conclusion in the Opinion that probability of CBS establishment in the EU is moderately likely is not affected by the paper by Magarey et al. (2015) predicting establishment in some of the EU locations they selected. The high level of uncertainty regarding the probability of establishment is also unchanged by Magarey et al. (2015). The Panel concluded that Martinez-Minaya et al. (2015) does not provide new evidence requiring an update to EFSA PLH Panel (2014), principally because it had already been concluded that global climate zones are based on factors and thresholds that are broad and not necessarily representative of those that are critical for the pathogen and its host.

This opinion addresses the question of the efficacy of current treatment solutions to cure Xylella fastidiosa diseased plants, and discusses the experimental treatments under evaluation by two research groups in Apulian olive orchards infected by strain CoDiRO (Complesso del Disseccamento Rapido dell’Olivo). The increasing problems from newly emerging vascular bacterial diseases and the limited success to cure plants from such infections have stimulated numerous studies on treatments with chemical and biological compounds. Under field conditions, various formulations of copper and zinc as spray or root drench are currently used while further options, for example the application of bioactive substances, are at an experimental stage. In Apulia, preliminary results from intensive treatments with such formulations, in combination with the use of good crop management practices, reported more vigorous new growth of diseased trees. However, results provided so far confirmed the continued presence of X. fastidiosa after the treatments under evaluation. This is in agreement with current knowledge that there are no means to cure plants from this bacterial disease, in the sense of eliminating the pathogen from plant tissues. The reported positive response of the treated olive trees is most probably due to the effect of micronutrients and other bioactive compounds that, together with soil cultivation and agronomical practices, improve the vigour of the plants and their resilience to stress caused by bacterial infections. Notwithstanding the preliminary status of these findings, the Panel acknowledged the potentially positive effects of such treatments in prolonging the productive phase of olive trees and their putative relevance for the management of olive orchards, particularly in the containment area where eradication of the pathogen is considered no longer possible. The Panel also concluded that long-term studies are needed to confirm that the reported positive effects on crop performance can be sustained over many years.

Plant disease epidemics require plant pathogens to be dispersed to infect new hosts. Understanding dispersal is important for devising methods to improve detection and control of plant pathogens. This is not straightforward, as plant pathogens can be dispersed by air, rain, water or soil, and by vectors such as animals, pollen, various microbes, people and machinery and on infected plant material including seeds. Epidemics vary in time and space as a result of complex processes affecting inoculum availability and production, dispersal and survival processes, and also the coincidence of susceptible crop plants, which each interact with the weather. To reduce disease, exposure of crops to inoculum is often limited by separating crops in time and space, using crop rotation, including different varieties. A wide range of diagnostic methods are increasingly used to help with this by detecting plant pathogens before infection occurs to prevent introduction of exotic inoculum, or to improve applications of crop protection products. Key Concepts: Plant pathogens include fungi, protists (such as oomycetes and plasmodiophorids), bacteria, phytoplasmas, viruses and viroids. For plant disease epidemics to occur, these plant pathogens must be dispersed to infect new hosts. Plant pathogens can be dispersed by air, rain, water, soil, by animals, people and machinery and through infected plant material including pollen and seeds. A key method for crop protection is to limit dispersal by separating susceptible crops in time and space by crop rotation, use of different varieties, hygiene or management of inoculum and use of crop protection products (biologicals and pesticides) at key growth stages or when infection conditions are suitable. Some pathogens that are dispersed by air are adapted to survive freezing temperatures, high UV light levels and desiccation, whereas others only remain viable for short periods or in certain less extreme conditions. Some may even induce ice nucleation to promote rainfall to return them to the ground. Some plant pathogens are dispersed in rain-splash, usually short distances but occasionally longer when combined with strong winds. Pathogens can be dispersed in water films and groundwater or rivers. Insects and other invertebrates, pollen and protists may vector some pathogens, mainly viruses, viroids and phytoplasmas. One of the main reasons for new introductions of plant pathogens to a territory is introduction by people, as infected plant material, infected seeds or as contamination on clothing, machinery, food or other imported materials. Various statutory quarantine regulations and inspections by plant health professionals aim to reduce introductions. Climate change may help pathogens to establish in new territories where they were previously absent. Keywords: epidemics; plant disease; plant pathogens; inoculum; aerobiology; rain-splash; soilborne; diagnostics; quarantine

BACKGROUNDMethyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in -tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESUL P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. MBC-target encoding gene -tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with -tubulin amino acid substitutions L240F and E198A. MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations. (c) 2013 Society of Chemical Industry

The colonization of winter oilseed rape plants and epidemiology of phoma stem canker differed between A/Tox(+) and B /Tox(0) Leptosphaeria maculans. In France and England, where plant colonization was investigated during two and three growing seasons, respectively, there was a difference in timing of leaf infection; A/Tox(+) L. maculans was predominant on leaves in the autumn (October/ November) but there was an increase in the incidence of B/Tox(0) in the winter (January/ February). In May, June and July both species could be isolated from all external parts of the plant (root to the upper stem) and all crown (stem base) tissues, although they differed in their distribution. At the root and crown, A/Tox(+) L. maculans was predominant and was located throughout the cortex, wood and pith tissues, but the rarer B/Tox(0) was located mainly in the cortex. Approximately equal numbers of A/Tox(+) and B/Tox(0) isolates were obtained from the upper stem - there was a greater proportion of B/Tox(0) isolates than at the crown. In England, after harvest in 1999 and 2000, pseudothecia on the lignified tap root and crown tissues produced predominantly A/Tox(+) ascospores (94%), while pseudothecia higher up the stem produced more B/Tox(0) ascospores (60%) than A/Tox(+) ascospores (40%). The timing of the onset of leaf spotting, earlier in the season for A/Tox(+) than B/Tox(0) L. maculans, and the predominance of mycelium of A/Tox(+) at the crown are consistent with the assumption that A/Tox(+) is more likely to cause the most damaging stem cankers than B/Tox(0) L. maculans. Identification as A/Tox(+) or B/Tox(0) by cultural characteristics differed only slightly (2.3%) from identification by molecular techniques.

In controlled-environment experiments, ascospores of both A-group and B-group Leptosphaeria maculans were able to infect leaves of oilseed rape and produce phoma leaf spot lesions at temperatures from 5 to 20 degreesC and wetness durations from 8 to 72 h after inoculation. Lesions formed on leaves inoculated with B-group ascospores had few pycnidia and were darker, smaller and less noticable than the larger, pale grey lesions with many pycnidia produced by A-group ascospores. Lesions formed by A-group or B-group L. maculans on naturally infected winter oilseed rape experimental crops were similar to lesions produced by the two groups on inoculated plants. The greatest numbers of lesions were produced with a leaf wetness duration of 48 h and at temperatures of 15-20 degreesC for both A-group and B-group ascospores. As leaf wetness duration and temperature decreased below the optimal values, the number of lesions decreased. The incubation period, estimated as the time from inoculation to the appearance of the first lesions (t(1)), or the time to the appearance of 50% of the lesions (t(50)), of B-group was often shorter than that of A-group L. maculans. As temperature decreased below 20 degreesC, the length of the incubation period of both A-group and B-group L. maculans increased.