Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index,X. diversicaudatum,X. vuittenezi and X. italiae,and for the quantification of X. index numbers

The ectoparasitic dagger nematodes Xiphinema index and Xiphinema diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real‐time PCR, for the specific detection of these species and of the closely related non‐vector species Xiphinema vuittenezi and Xiphinema italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species whatever the nematode population. The specificity of detection and absence of false positive reaction were confirmed in samples of each species mixed with the three other Xiphinema species or mixed with nematodes representative from other genera (non‐plant‐parasitic Dorylaimida, Longidorus sp., Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. index numbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time‐consuming microscopic identification and counting, this real‐time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.     

Effect of cutting frequency on above‐ and belowground biomass production of Rumex alpinus,R. crispus,R. obtusifolius and the Rumex hybrid (R. patienta × R. tianschanicus) in the seeding year

Rumex species are important weeds in grasslands and on arable land. The Rumex hybrid (R. patienta × R. tianschanicus; cv. OK‐2, Uteusha) has been planted as a forage and energy crop since 2001 in the Czech Republic, but its ecological requirements and its potential to become a new weedy species have never been investigated. In 2010 and 2011, we performed a pot experiment to investigate the effect of none, one and two cuts per year on biomass production of Rumex OK‐2 and common broad‐leaved Rumex species (Rumex obtusifolius, R. crispus and R. alpinus). The higher cutting frequency can reduce the belowground biomass, but no effect on the aboveground biomass was detected. Flowering in the seeding year was recorded in only 50% of R. obtusifolius plants. Non‐flowering R. obtusifolius plants produced significantly more belowground biomass than flowering plants under no cutting or one cut treatments. The growth response of Rumex OK‐2 to different cutting treatments was very similar to R. crispus. These similarities indicate the weed potential of the hybrid to become a troublesome weedy species, similar to R. crispus.     

Mechanisms of resistance in Brassica carinata,B. napus and B. juncea to Pseudocercosporella capsellae

Studies were undertaken to compare susceptible and resistant host responses to Pseudocercosporella capsellae in cotyledons of Brassica carinata, B. juncea and B. napus in order to define the mechanisms of resistance in these three species. On both resistant and susceptible hosts, hyphal penetration was always through stomatal openings and without infection pegs or appressoria. On resistant B. carinata ATC94129P, up to 72% of spores disintegrated and, generally, germination (<22%) and germ tube lengths (<25 μm) were comparatively low. Resistant B. napus Hyola 42 had the lowest germination (8%) and susceptible B. carinata UWA#012 had the highest (51%). On resistant B. carinata ATC94129P, germ tube extension was impeded across 24–60 h post‐inoculation (hpi) and percentage stomatal penetration lower (4%) at 60 hpi compared with susceptible B. carinata UWA#012 (26%). Stomatal densities (stomata/14 757 μm²) on resistant B. juncea Dune (2·12) and B. napus Hyola 42 (1·62) were lower than for susceptible B. juncea Vardan (2·40) and B. napus Trilogy (2·03). Resistant B. carinata ATC94129P had greater stomatal density (1·89) than susceptible B. carinata UWA#012 (1·58). Overall, B. juncea had greater stomatal density (2·26) compared with B. napus (1·83) and B. carinata (1·74). In resistant B. carinata ATC94129P, P. capsellae induced 28% stomata to close, while in susceptible B. carinata UWA#012 no such closure was induced. Epicuticular wax crystalloids were present only on resistant B. carinata ATC94129P and probably also contribute towards resistance.     

Epidemiological importance of Solanum sisymbriifolium, S. nigrum and S. dulcamara as alternative hosts for Phytophthora infestans

Lesions of Phytophthora infestans were found on woody nightshade ( Solanum dulcamara ), black nightshade ( S. nigrum ) and S. sisymbriifolium during a nationwide late blight survey in the Netherlands in 1999 and 2000. Pathogenicity and spore production of P. infestans isolates collected from potato ( S. tuberosum ), S. nigrum , S. dulcamara and S. sisymbriifolium were determined on several host plant species, and oospore formation in naturally infected and inoculated foliage of hosts was quantified. The present population of P. infestans in the Netherlands is pathogenic on S. nigrum , S. dulcamara and S. sisymbriifolium . Oospores were produced in leaves of S. nigrum , S. dulcamara and S. sisymbriifolium following infection with A1 and A2 isolates. Therefore these plant species should be regarded as alternative hosts for the late blight pathogen. In the case of S. nigrum and S. dulcamara infection was a relatively rare event, suggesting that diseased plants do not significantly contribute to the overall late blight disease pressure present in potato-production areas. Oospore production in ageing S. nigrum and S. dulcamara plants in autumn, however, may generate a considerable source of (auto) infections in following years. Considerable numbers of sporangia and oospores were produced on S. sisymbriifolium following infection with P. infestans . Additional field infection data are needed to evaluate the epidemiological consequences of a commercial introduction of S. sisymbriifolium as a potato cyst nematode trap crop.     

Effect of electrical conductivity on survival of Phytophthora alni,P. kernoviae and P. ramorum in a simulated aquatic environment

This study investigated survival of the pathogens Phytophthora ramorum, P. alni and P. kernoviae as zoospores or sporangia in response to an important water quality parameter, electrical conductivity (EC), at its range in irrigation water reservoirs and irrigated cropping systems. Experiments with different strengths of Hoagland’s solution showed that all three pathogens survived at a broad range of EC levels for at least 3 days and were stimulated to grow and sporulate at ECs > 1·89 dS m^?1. Recovery of initial populations after a 14‐day exposure was over 20% for P. alni subsp. alni and P. kernoviae, and 61·3% and 130% for zoospores and sporangia of P. ramorum, respectively. Zoospore survival of these pathogens at ECs < 0·41 dS m^?1 was poor, barely beyond 3 days in pure water; only 0·3% (P. alni), 2·9% (P. kernoviae) and 15·1% (P. ramorum) of the initial population survived after 14 days at EC = 0·21 dS m^?1. The variation in rates of survival at different EC levels suggests that these pathogens survive better in cropping systems than in irrigation water. Containment of run‐off and reduction in EC levels may therefore be non‐chemical control options to reduce the risk of pathogen spread through natural waterways and irrigation systems.     

Pathogenicity of Hymenoscyphus fraxineus towards leaves of three European ash species: Fraxinus excelsior,F. angustifolia and F. ornus

This study aimed to demonstrate the association of the ash dieback pathogen Hymenoscyphus fraxineus with leaf symptoms on Fraxinus excelsior and to test its pathogenicity towards leaves of three European ash species, F. excelsior, F. angustifolia and F. ornus, in wound inoculation experiments. On F. excelsior, H. fraxineus was isolated from 94% of leaf rachises with necrotic lesions and from 74% of necrotic leaflet midribs. Following wound inoculation of leaf rachises, in two separate experiments performed in 2010 and 2011, the ash dieback pathogen caused symptoms (necrotic rachis lesions, leaf wilting and premature leaf shedding) on all three ash species, while control leaves remained symptomless. Hymenoscyphus fraxineus was consistently reisolated from fungus‐inoculated rachises. All 10 isolates tested were pathogenic to the three ash species and varied in virulence. Koch's postulates for H. fraxineus as causal agent of leaf symptoms on F. excelsior were fulfilled in this study. Complemented with the isolation of the fungus from naturally infected, symptomatic leaf rachises of F. angustifolia and F. ornus in previous investigations, H. fraxineus was confirmed to be a leaf pathogen of these ash species as well. The leaf inoculation experiments showed that F. excelsior was highly susceptible to H. fraxineus, F. angustifolia was equally or slightly less susceptible, whereas F. ornus was the least affected species; however, F. ornus should also be regarded as a host tree for the ash dieback pathogen. This susceptibility ranking corresponds well with field observations and previous stem inoculation experiments.     

Clavibacter michiganensis subsp. sepedonicus

Specific scope

This standard describes a national regulatory control system for Clavibacter michiganensis subsp. sepedonicus that provides guidance on surveillance for the pathogen and its containment and eradication if found.

Specific approval and amendment

First approved in 2003–09. Revision approved in 2011–09.     

Colonization of lettuce cultivars and rotation crops by Fusarium oxysporum f. sp. lactucae,the cause of fusarium wilt of lettuce

The severity of fusarium wilt is affected by inoculum density in soil, which is expected to decline during intervals when a non‐susceptible crop is grown. However, the anticipated benefits of crop rotation may not be realized if the pathogen can colonize and produce inoculum on a resistant cultivar or rotation crop. The present study documented colonization of roots of broccoli, cauliflower and spinach by Fusarium oxysporum f. sp. lactucae, the cause of fusarium wilt of lettuce. The frequency of infection was significantly lower on all three rotation crops than on a susceptible lettuce cultivar, and the pathogen was restricted to the cortex of roots of broccoli. However, F. oxysporum f. sp. lactucae was isolated from the root vascular stele of 7·4% of cauliflower plants and 50% of spinach plants that were sampled, indicating a greater potential for colonization and production of inoculum on these crops. The pathogen was also recovered from the root vascular stele of five fusarium wilt‐resistant lettuce cultivars. Thus, disease‐resistant plants may support growth of the pathogen and thereby contribute to an increase in soil inoculum density. Cultivars that were indistinguishable based on above‐ground symptoms, differed significantly in the extent to which they were colonized by F. oxysporum f. sp. lactucae. Less extensively colonized cultivars may prove to be superior sources of resistance to fusarium wilt for use in breeding programmes.     

SIMPEROTA 1/3 – a decision support system for blue mould disease of tobacco*

Blue mould (Peronospora tabacina) is the most serious threat to German tobacco crops. In order to efficiently control the disease whilst minimizing the risk of nontolerable fungicide residue levels on tobacco leaves, a decision support system has been developed which optimizes the timing of fungicide treatments. The DSS consists of two models, SIMPEROTA 1, which forecasts the dates of blue mould first appearance and SIMPEROTA 3 which forecasts the dates of fungicide applications. Crucial biological processes are included in the models (infection, mycelium growth, sporulation and spore release). Input parameters are temperature, relative humidity and leaf wetness recorded on an hourly basis. Validation with data from 2003 and 2006 showed that SIMPEROTA 1 gave satisfying results. The model is suitable for practical use and can be employed for steering monitoring efforts of extension services and for the timing of the first fungicide treatment. SIMPEROTA 3 gives advice on follow‐up treatments and the length of spraying intervals, but this model needs to be validated before being introduced into practice.     

PM 7/21 (2) Ralstonia solanacearum,R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex)

Specific scope

This Standard describes a diagnostic protocol for Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii, i.e. phylotype/sequevar strain in the Ralstonia solanacearum Species Complex (RSSC). 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.

Specific approval and amendment

Approved in 2003‐09. First revised in 2018‐02.     

Unique infection structures produced by Pseudocercosporella capsellae on oilseed crops Brassica carinata,B. juncea and B. napus in Western Australia

White leaf spot disease (Pseudocercosporella capsellae) is widespread across oilseed, vegetable and forage brassicas. Light (LM) and scanning electron (SEM) microscope studies were undertaken to investigate host–pathogen interactions on cotyledons of resistant and susceptible Brassica carinata, B. juncea and B. napus. Under LM, unique brown structures were present, particularly on susceptible genotypes, in two morphologically distinct forms: first, as thread‐like structures within cortical tissue by 24 h post‐inoculation (hpi) and secondly, as brown ropy strand structures either within cortical tissues (internal ropy strands), or extruded out through stomatal pores (ropy strand extrusions). Under LM, these brown structures were most prevalent in highly susceptible B. juncea ‘Vardan’ that had both a high incidence within cortical tissue (70%) and of ropy strand extrusions (73%), as did susceptible B. napus ‘Trilogy’ within cortical tissue (60%). Under SEM, both these genotypes showed thread‐like structures smaller than hyphae forming highly branched networks and ropy strand‐like structures. While there were fewer brown structures in susceptible B. carinata UWA #012 (35%), fine, thread‐like structures forming networks were again prominent (SEM). In contrast, for resistant genotypes, brown structures (LM) were of very low frequency or absent; only 5% in resistant B. juncea ‘Dune’ and none in resistant B. napus ‘Hyola 42’ or highly resistant B. carinata ATC94129P. Under SEM, fine, thread‐like structures were present in the resistant B. juncea ‘Dune’ and B. napus ‘Hyola 42’. Liquid chromatographic analyses of brown structures revealed that both internal ropy strands within cortical tissues and ropy strand extrusions contained the mycotoxin cercosporin.     

Relationship between Spongospora subterranea f. sp. subterranea soil inoculum level,host resistance and powdery scab on potato tubers in the field

The relationship between initial soil inoculum level of Spongospora subterranea f. sp. subterranea (Sss) and the incidence and severity of powdery scab on potato tubers at harvest was investigated. In all experiments soil inoculum level of Sss (sporeballs/g soil) was measured using a quantitative real‐time PCR assay. Of 113 commercial potato fields across the UK, soil inoculum was detected in 75%, ranging from 0 to 148 Sss sporeballs/g soil. When arbitrary soil inoculum threshold values of 0, <10 and >10 sporeballs/g soil were set, it was observed that the number of progeny crops developing powdery scab increased with the level of inoculum quantified in the field soil preplanting. In four field trials carried out to investigate the link between the amount of inoculum added to the soil and disease development, disease incidence and severity on progeny tubers was found to be significantly (P < 0·01) greater in plots with increasing levels of inoculum incorporated. There was a cultivar effect in all years, with disease incidence and severity scores being significantly greater in cvs Agria and Estima than in Nicola (P < 0·01).     

Pathogenic and non‐pathogenic Agrobacterium tumefaciens,A. rhizogenes and A. vitis strains form biofilms on abiotic as well as on root surfaces

Pathogenic and non‐pathogenic Agrobacterium tumefaciens, A. rhizogenes and A. vitis strains growing in minimal liquid medium adhered to different abiotic surfaces, forming biofilms at initial stages of development. Agrobacterium tumefaciens and A. vitis strains were able to attach to both polystyrene and polypropylene materials, whereas the A. rhizogenes strains only bound to polystyrene surfaces. Strains of the three species were also able to form biofilms on borosilicate coverslips. It is concluded that their ability to adhere to and form nascent biofilms on abiotic surfaces is dependent on the Agrobacterium species (biovar), surface material and growth conditions. Furthermore, tumorigenic A. tumefaciens and A. vitis strains, and the biological control agent A. rhizogenes strain K84, bound tightly to and formed complex biofilms on the surface of tomato root tips ex planta. More importantly, in planta assays confirmed that all three Agrobacterium spp. strains efficiently colonized tomato seedlings and also formed biofilms on roots. These complex structures, as revealed by scanning electron microscopy, were composed of numerous bacterial cells arranged in different ways: either dense and continuous carpets, large aggregates embedded in extra‐cellular material or globular mushrooms traversed internally by channels. Confocal laser scanning microscopy, using GFP‐marked derivative strains, corroborated the presence of live, three‐dimensional and thick green fluorescent structures attached to plant material. This study illustrates that besides A. tumefaciens, strains of the species A. rhizogenes and A. vitis are also able to build biofilms on abiotic as well as on root surfaces.     

Allelopathic Potential of Soliva sessilis Ruiz & Pav. on Wheat

Soliva sessilis (Burweed) is a winter annual weed found in wheat crop of Pakistan. It is thought to have an allelopathic effect on wheat crop. In this regard, an experiment was conducted in 2019 in the department of Weed Science and Botany at the University of Agriculture Peshawar, to assess the allelopathic potential of S. sessilis on various wheat varieties grown in Peshawar, Pakistan. Physiologically matured roots and leaves of S. sessilis were dried, ground and stored. Three common wheat varieties were selected for the research including Atta Habib, Pirsabaq-15, and Shahkar-13. The treatments of the experiment comprised of the aqueous extracts of the roots and leaves of S. sessilis, hot water extracts, methanol extracts, litter, and mulches. A mass of 5?g of S. sessilis was taken for the different extracts for 24?h, however, the methanol extract was kept for 48?h. The effect of the treatments was highly significant on wheat seed germination, radicle length, plumule length, fresh biomass as well as dry biomasses of the three tested wheat varieties. The variety Atta Habib was discovered as the most susceptible variety to the treatments of aqueous extracts, hot water extracts, and leaves’ litter. In contrary, the variety Pirsabaq-15 exhibited a remarkable increase in the various data collection parameters. The leaves of S. sessilis were observed more toxic as compared to the roots. The mulch of S. sessilis indicated stimulatory effect on the wheat varieties. For the interaction effect between treatments and varieties, the leaves and roots aqueous extract and their litter showed inhibitory effect. In conclusion, the leaves and roots of S. sessilis are potentiality allelopathic to wheat crop plants and further study is needed to assess the types of these allelochemicals present in the leaves and roots of the weed.