Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

Three nursery fields and three rootstock mother fields from commercial nurseries located in Comunidad Valenciana region (central‐eastern Spain) were surveyed in July 2011 to detect the presence and to quantify Ilyonectria spp. in the soil. In each field, ten soil samples were taken randomly with a soil probe at a depth of 10–30 cm, and 10–20 cm from the base of the plant. Three replicate subsamples (10 g each) were taken from each soil sample. DNA was extracted and a multiplex nested PCR with species‐specific primer pairs (Mac1/MaPa2, Lir1/Lir2 and Pau1/MaPa2) was used to identify the species present. Among the 180 soil DNA samples analysed, Ilyonectria spp. were detected in 172 of them. Ilyonectria macrodidyma complex was the most frequently detected, being identified in 141 samples from all the fields evaluated. However, I. liriodendri was detected in only 16 samples, but was present in all open‐root field nurseries and in two rootstock mother fields. In addition, quantitative PCR (qPCR) assays were done to assess the levels of I. liriodendri and I. macrodidyma‐complex DNA in the soil samples. Detection of Ilyonectria spp. DNA using qPCR correlated with the fields found positive with the nested multiplex PCR. DNA concentrations of Ilyonectria spp. ranged from 0·004 to 1904·8 pg μL^?1. In general, samples from rootstock mother fields showed the highest DNA concentrations. The ability to detect and quantify Ilyonectria spp. genomic DNA in soil samples from nursery fields and rootstock mother fields confirms soils from both field types as important inoculum sources for black‐foot pathogens.     

High genetic and virulence diversity detected in Neofusicoccum luteum and N. australe populations in New Zealand vineyards

Genotypic and virulence diversity of Neofusicoccum luteum and N. australe isolates recovered from grapevines displaying symptoms of dieback and decline in New Zealand were investigated. The universally primed PCR (UP‐PCR) method was used to investigate the genetic diversity of 40 isolates of N. luteum and 33 isolates of N. australe. Five UP‐PCR primers produced a total of 51 loci from N. luteum and 57 from N. australe with a greater number of polymorphic loci produced in N. australe (86%) compared with N. luteum (69%). Analysis of UP‐PCR data showed both species found in New Zealand vineyards were genetically diverse at both the inter‐ and intra‐vineyard levels with only a single pair of clonal isolates in N. luteum. Cluster analysis of UP‐PCR data produced four genetic groups in N. luteum and 10 in N. australe (P < 0.05). For both species, there was no relationship between the genetic groups and the origin of isolates. The mean genetic diversity (H) of N. luteum was less than for N. australe, being 0.1791 and 0.2417, respectively. Pathogenicity assays of both species using isolates from either the same or different genetic groups inoculated onto either green shoots or grapevine trunks, showed virulence diversity within the population; however, no correlation was identified between genetic groups and virulence.     

Development of a grower‐conducted inoculum detection assay for management of grape powdery mildew

Management of grape powdery mildew (Erysiphe necator) and other polycyclic diseases often relies on calendar‐based pesticide application schedules that assume the presence of inoculum. An inexpensive, loop‐mediated isothermal amplification (LAMP) assay was designed to quickly detect airborne inoculum of E. necator to determine when to initiate a fungicide application programme. Field efficacy was tested in 2010 and 2011 in several commercial and research vineyards in the Willamette Valley of Oregon from pre‐bud break to véraison. In each vineyard, three impaction spore traps were placed adjacent to the trunk. One trap was maintained and used by the grower to conduct the LAMP assay (G‐LAMP) on‐site and the other two traps were used for laboratory‐conducted LAMP (L‐LAMP) and quantitative PCR assay (qPCR). Using the qPCR as a gold standard, L‐LAMP was comparable with qPCR in both years, and G‐LAMP was comparable to qPCR in 2011. Latent class analysis indicated that qPCR had a true positive proportion of 98% in 2010 and 89% in 2011 and true negative proportion of 96% in 2010 and 64% in 2011. An average of 3·3 fewer fungicide applications were used when they were initiated based on spore detection relative to the grower standard practice. There were no significant differences in berry or leaf incidence between plots with fungicides initiated at detection or grower standard practice plots, suggesting that growers using LAMP to initiate fungicide applications can use fewer fungicide applications to manage powdery mildew compared to standard practices.     

Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39‐ and BTH‐activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH‐activated resistance was consistently high in the different cultivars. Expression profiles of defence‐related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence‐related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence‐related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.     

Use of nitrate non‐utilizing (nit) mutants to determine phenological stages at which Botrytis cinerea infects wine grapes causing botrytis bunch rot

Botrytis bunch rot (BBR), caused by Botrytis cinerea, degrades wine grapes during ripening, even though infection can occur as early as flowering. Effective BBR management requires knowledge of whether some stages of fruit development are more important than others in relation to infection and BBR severity at harvest. Bunches of Vitis vinifera ‘Sauvignon blanc’ and/or ‘Pinot noir’ were inoculated in two vineyard trials and one glasshouse trial with nitrate non‐utilizing (nit) mutant strains at three phenological stages: early flowering, pre‐bunch closure (PBC) and veraison. Isolates recovered from symptomless berries at veraison and from bunches with symptoms at harvest were screened to measure the incidence of the nit strains used in the inoculations. It was found that latent infections, which resulted in BBR at harvest, could become established at all three phenological stages and no single stage was associated with greater latent incidence or harvest severity than any other stage. It was concluded that a proportion of BBR at harvest resulted from the expression of latent infections that had accumulated throughout the season. However, the time between infection and BBR symptom expression in near‐ripe grape berries was sufficiently short for polycyclic secondary infection to also contribute to epidemic development.     

Intraspecific variation in Diplodia seriata isolates occurring on grapevines in Spain

Variation of Diplodia seriata, a fungal species associated with botryosphaeria dieback of grapevine, was investigated with respect to its genetic, phenotypic and pathogenic characteristics. The inter‐simple sequence repeat (ISSR) technique was used to investigate the genetic diversity of 83 isolates of D. seriata. Five ISSR primers were able to provide reproducible and polymorphic DNA fingerprint patterns, thus showing a relevant genetic variability in the species. Analyses of ISSR data by different clustering methods grouped the isolates into two distinct clusters through the Bayesian and DAPC analyses. No relationships between either geographic or host origin of isolates and genetic clusters were observed. Several representative isolates from each genetic cluster were chosen for studying their conidial dimensions, in vitro mycelial growth, vegetative and mating compatibility, and pathogenicity on detached grapevine canes and potted vines. No significant differences in conidial dimensions were detected among the groups. Vegetative compatibility reactions were observed among isolates but this was not related with the genetic clustering. Production of sexual fruiting bodies in vegetative compatible crossings was not observed under the experimental conditions used in the study. All 14 isolates tested for pathogenicity were confirmed to be pathogenic according to the length of the necrotic lesions that they caused and their reisolation frequencies from the infected plant tissues. Differences in the length of necrosis were detected among isolates, thus revealing the existence of different virulence levels in the species.     

A model for the development of Erysiphe necator chasmothecia in vineyards

A temperature‐driven, mechanistic model predicting the development of Erysiphe necator chasmothecia in vineyards was developed and validated in 38 vineyards in the Po Valley (northern Italy), Baden‐Württemberg (Germany), and South Australia between 2005 and 2011. The model, which begins operating when the first ascocarp initials are formed, predicts on a daily basis the proportions of chasmothecia at the yellow, brown and black maturity stage. The initialization date was estimated with an iterative procedure that minimized the residuals of predicted versus observed values. In all vineyards, a drop to more favourable temperatures for ascocarp production over 2–4 days in the week or in the 2 weeks before the model initialization date probably triggered chasmothecia production. Model predictions provided a good fit of observed data (coefficients of determination, model accuracy, efficacy and efficiency were all ≥0·90), with some overestimation. When predicted production of black chasmothecia (on leaves) was compared with observed dispersal of chasmothecia from vines, lack of splashing rain was probably the main cause of overestimation. When observed numbers of yellow, brown or black chasmothecia on leaves were compared with model predictions, removal of the developing chasmothecia by rainfall was probably the main cause of overestimation. Inclusion of the effect of rainfall on the removal of immature and mature chasmothecia from the powdery mildew colonies could improve the model. The model could be used to time the application of fungicides or biocontrol agents for reducing ascocarp formation and reducing primary inoculum in the following season.     

Genetic diversity of the root‐knot nematode Meloidogyne ethiopica and development of a species‐specific SCAR marker for its diagnosis

Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species‐specific SCAR‐PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species‐specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root‐knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.     

Role of co‐infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co‐infection of potato. The objective of this study was to determine if co‐infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real‐time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water‐soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co‐inoculation with these strains of V. dahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co‐inoculated with both pathogens compared to either pathogen alone in both trials, and V. dahliae levels were greater in basal stems of plants co‐inoculated with either Pectobacterium isolate. Overall, these results indicate that although co‐infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co‐infection can increase PED severity.     

Host switching between native and non‐native trees in a population of the canker pathogen Chrysoporthe cubensis from Colombia

The purpose of this study was to test the hypothesis that Chrysoporthe cubensis on native trees in South America could be the source of the pathogen that causes severe stem cankers and often mortality in commercially propagated Eucalyptus trees. This was done by investigating populations originating from two adjacent Eucalyptus (Myrtaceae) plantations in Colombia, and wild Miconia rubiginosa trees (Melastomataceae) growing alongside these stands. Polymorphic microsatellite markers were used to quantify allele sizes in 20 and 39 isolates from the two Eucalyptus stands and 32 isolates from adjacent M. rubiginosa trees. Gene and genotypic diversities were calculated from these data, and population differentiation and assignment tests were performed to ascertain whether the populations were genetically different. Results showed that there were no differences between any of the populations using these techniques, and that they can be treated as a single population. Therefore, the results support the hypothesis that host switching has occurred in C. cubensis in Colombia.     

Transmission of rice blast from seeds to adult plants in a non‐systemic way

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a serious threat to rice production worldwide. In temperate regions, where rice is not cultivated for several months each year, little is known about the initial onset of the disease in the field. The main overwintering and primary inoculum sources reported are infested residues and seeds, but the subsequent steps of the disease cycle are largely unknown, even though a systemic infection has been proposed but not demonstrated. The present work follows rice blast progression in infected seeds from germination to seedling stage, with direct and detailed microscopic observations under both aerobic conditions and water seeding. With the use of GFP‐marked M. oryzae strains, it was shown that spores are produced from contaminated seeds, infect emerging seedling tissues (coleoptile and primary root) and produce mycelium that colonizes the newly formed primary leaf and secondary roots. Using different rice cultivars exhibiting distinct levels of resistance/susceptibility to M. oryzae at the 2/4‐leaf stage, it was observed that resistance or susceptibility of a considered genotype is already established at the seedling stage. The results also showed that when plants are inoculated either at ripening stage (mature panicles), heading stage (flowering/immature panicles) or even before heading (flag leaf fully developed), they produce infested seeds. These seeds produce contaminated seedlings that mostly die and serve as an inoculum source for healthy neighbouring plants, which gradually develop disease symptoms on leaves. The possible rice blast disease cycle was reconstructed on irrigated rice in temperate regions.     

Development of a lateral flow device for in‐field detection and evaluation of PCR‐based diagnostic methods for Xanthomonas campestris pv. musacearum,the causal agent of banana xanthomonas wilt

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in‐field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross‐reacted with non‐target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 10⁵ cells mL^?1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first‐line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non‐scientists and is cost‐effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.     

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.     

Selectivity of foramsulfuron + thiencarbazone‐methyl and classic herbicides in sensitive and non‐sensitive sugar beet genotypes

A new herbicide for sugar beet cultivation using the ALS‐inhibiting active ingredients foramsulfuron and thiencarbazone‐methyl is under approval in the EU member states. Sugar beet genotypes that are non‐sensitive to this herbicide are currently under development. Selectivity of the ALS‐inhibiting herbicide and yield response of the non‐sensitive genotypes might be relevant to meet the requirements for variety registration. To evaluate these issues, six field trials were conducted in Germany in 2013 and 2014. Classic herbicides and the ALS‐inhibitor herbicide were applied in dosages of up to fourfold the authorised (or applied for) application rates. The ALS‐inhibitor herbicide did not cause any significant phytotoxicity and had no effect on leaf area index at a single, double or fourfold dosage. By contrast, classic herbicides had significant negative effects at the single dosage. At fourfold dosage, they caused 41% phytotoxicity and reduced leaf area index by 35%. The relative yield difference between ALS‐inhibitor and classic herbicide treatments was 8.6% and 17.4% of white sugar yield at double and fourfold dosage respectively. The ALS‐inhibitor herbicide thus showed higher selectivity than the classic herbicides. In the registration process, the resulting yield advantage could balance a possible yield penalty of non‐sensitive genotypes. The introduction of a new system for weed control could improve application flexibility and control of troublesome weeds in sugar beet.     

Quantification of Mirafiori lettuce big‐vein virus and its vector,Olpidium virulentus,from soil using real‐time PCR

Big vein disease of lettuce (Lactuca sativa) is an economically important disease transmitted through soil by Olpidium virulentus, and has occurred in most production areas worldwide. The disease is assumed to be caused by Mirafiori lettuce big‐vein virus (MiLBVV). To understand the dynamics of the virus and its vector, MiLBVV and O. virulentus were directly detected in soil. DNA and RNA were extracted from 5 g soil using a bead beating method, followed by purification using adsorption to a column. Detection and quantification were performed using real‐time PCR and a TaqMan probe that was prepared based on the CP region of MiLBVV and the rDNA‐ITS region of O. virulentus, respectively. Furthermore, using a visual assessment of the incidence rate of big vein disease on lettuce in agricultural fields, the C_t values of MiLBVV and O. virulentus from soil were also determined using real‐time PCR. The results showed that MiLBVV concentrations in the soil were high in the field, as also determined by a visual assessment of the incidence rate of big vein disease on lettuce. However, the amount of O. virulentus in soil was not directly correlated with the incidence of MiLBVV. From these results, it is suggested that the risk of lettuce crops developing big vein disease can be estimated using an index of the amount of MiLBVV in the soil.