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The main and most efficient measure to control potato cyst nematode (PCN) is the use of resistant cultivars. German and Dutch National Plant Protection Organizations (NPPOs) recently reported the emergence of Globodera pallida populations virulent on potato cultivars carrying resistance against pathotype 2/3. The development and virulence of the virulent population Oberlangen from Germany in comparison to the reference population G. pallida Pa3 Chavornay were investigated on resistant and susceptible cultivars in glasshouse experiments. Various life history traits associated with change in virulence were also assessed. Hatching of second-stage juveniles (J2s) was similar for both populations but incubation of cysts in potato root diffusate resulted in higher hatching rates compared to 3 mM Zinc Chloride and tap water. Both populations showed high penetration rates in the roots of the resistant and susceptible cultivars. However, only the population Oberlangen was able to complete the life cycle in the roots of the resistant potato cultivar. In ‘Seresta’, the resistance response restricted the formation of females by avirulent individuals in favor of males. Oberlangen was virulent on all cultivars tested. No difference in cyst size, number of eggs per cyst, length of juveniles, and males was found for Oberlangen and Chavornay on the susceptible cv. ‘Desiree’. However, cysts of virulent populations from the same region Oberlangen was obtained from had a significantly larger diameter compared to avirulent populations. The population Oberlangen showed a higher reproduction and fitness than the reference population Chavornay on susceptible cultivars and could serve as a future reference population in testing of new potato cultivars for resistance against this new virulence type in Europe.

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Summary: The stem nematode, Ditylenchus dipsaci , causes severe damage in sugar beet. To date, nematode inoculation through the leaf axil has been used as the standard method to investigate D. dipsaci interaction with sugar beet under in vivo conditions. To get as close as possible to field conditions, we established a new screening mechanism to perform soil inoculation. The most suitable inoculation time point, inoculum level and positioning on sugar beet, as well as rearing process on carrots, were determined. At a 15:8°C day:night temperature regime, penetration rates of D. dipsaci were at maximum following soil inoculation at plant emergence. Up to 115 nematodes penetrated sugar beet seedlings 22 days post-planting with an inoculum level of 1000 nematodes into the soil at plant emergence. Ditylenchus dipsaci penetration rate was higher in plants with soil inoculation than with inoculation on to the leaf axil. High soil moisture increased nematode migration into seedlings when D. dipsaci inoculation was carried out in four holes 1 cm from the plant base. Rearing the nematodes for 35 days at 20°C on carrot discs resulted in an infective inoculum containing up to 50% eggs. We recommend a soil inoculation of 1000 freshly extracted nematodes per pot at plant emergence. The nematode suspension has to be previously reared for 35 days on carrot discs to obtain active D. dipsaci inoculum. This system will allow for the selection of suitable sugar beet genotypes that suppress nematode penetration, in support of breeding for resistance against D. dipsaci. [ABSTRACT FROM AUTHOR]

Summary: Population-level genetic tools to study the epidemiology of potato cyst nematodes (PCN) are needed to evaluate their adaptive potential and validate management strategies. The effector gene vap1 is essential in early infection by PCN. We expected high allelic variation due to diversifying selection. A technique was developed specifically to amplify vap1 fragments of PCN, and electrophoretically separate variants in a denaturing gradient (DGGE). Sequenced gene variants differed in intron as well as exon regions. Siblings were more similar in vap1 pattern than juveniles from different cysts. Nearly all juveniles analysed had unique patterns with up to ten or five vap1 variants for Globodera pallida or G. rostochiensis , respectively. Patterns differed significantly among populations, with similarities increasing from between-species, within-species to within-pathotype comparisons. In conclusion, the method is suitable to determine the similarity of heterogeneous PCN populations for studies on their temporal change and spread. [ABSTRACT FROM AUTHOR]

Summary: Globodera pallida infestation on potato is responsible for huge yield losses globally. Screening of potato germplasm for resistance to the nematode at the early stages of a breeding programme can significantly enhance resistance-based management. This study assessed the suitability of tissue culture (TC)-derived potato plants as screening material for resistance to G. pallida. Reproduction of the nematode on TC plants was similar to the reproduction on tuber- and eye-plug-derived plants. The pot volume, inoculum density and inoculation time had a significant effect on the reproduction. A positive correlation was found between the mean number of white females on the root surface and the final number of nematode cysts after extraction. Resistance ranking using TC plants and the tubers yielded comparable results, thus justifying the use of TC in the screening process. Tissue culture plants have the potential of speeding up the screening process and reducing resource requirements, thus lowering breeding cost. [ABSTRACT FROM AUTHOR]

Plant-parasitic nematodes can cause significant damage to agricultural crops and forests worldwide, resulting in major economic losses. Some nematode species do not occur in all areas and are regulated as quarantine organisms. To avoid introduction and spread of these organisms, fast, simple and reliable detection and identification methods are needed, that help plant diagnostic services such as reference centres or national plant protection organizations (NPPOs) to rapidly identify suspicious nematodes. Real-time PCR is one of the fastest, most sensitive and reliable methods to fulfil this task. It is a DNA-based method that is easy to learn with the only requirement of having a specific thermocycler (Real-time Platform) and the appropriate chemistry. Real-time PCR provides very sensitive detection and species-specific identification with the potential to quantify target organisms if required. Following DNA extraction, results can be seen in 1-3 h and management decisions applied. Real-time PCR can be used for high-throughput analysis of many samples and in some cases for multiplexing, allowing for identification of more than one species in a single reaction. Over the past 15 years, real-time PCR methods have been developed for the main plant-parasitic nematodes, in particular the regulated species. This paper reviews the achievements in plant nematology diagnostics using real-time PCR as the method of choice for fast and reliable detection, identification and even quantification of plant parasitic nematodes. [ABSTRACT FROM AUTHOR]

The emergence of the new virulence type "Emsland" of the quarantine potato cyst nematode Globodera pallida (G. pallida) has led to a new thread for potato production. Since there are neither resistant potato cultivars against the new virulence type nor nematicides available, a sufficient monitoring is crucial. Once a field is contaminated with G. pallida, the nematode is able to endure 20 years or longer in the field, which blocks future cultivation of potatoes or other Solanaceae in the crop rotation. The new virulent population was able to spread undetected, due to cost- and labor-intensive conventional monitoring systems. The typical occurrence in nests of cyst nematodes reduces the probability of localizing an infestation, in addition to the high effort for soil sampling and extraction for detection and identification. If an infestation is recognizable, for example by wilting symptoms, stunted growth or a positive result after taking a soil sample, an area is already significantly contaminated. The objective of the research project is to develop a detection system based on hyperspectral sensor information. The target value is the detection of early/latent infestations. An early/latent infestation already leads to a strong increase in population densities of G. pallida, without visible symptoms. In addition, the level of virulence present should be estimated. The detection system will be based on an imaging hyperspectral camera (400 -- 1000 nm) and on a leafclip attachment, at different inoculum levels of G. pallida. Additionally, developmental stages are investigated by destructive sampling in combination with determination of water, chlorophyll and nitrogen content of the plants. The collected data should be incorporated into a self-learning algorithm, to support early detection of a latent G. pallida infestation of virulent populations. The project aims to develop detection methods for virulent G. pallida populations and provides the groundwork for monitoring systems, e.g. using drones or remote sensing. The results serve as a basis for the plant protection services to protect production areas from long-term contamination and thus to avoid a long-term blocking of potato cultivation. [ABSTRACT FROM AUTHOR]

Meloidogyne enterolobii, is one of the economically important root-knot species as its ability to develop and reproduce on host plants carrying resistance to other major root-knot nematodes. Recent reports around the world on wide range of plants species including major crops depicts its wide spread. Due to polyphagous nature of M enterolobii and limited control measures, it is important to understand host range to reduce further spread and develop control measures. Meloidogyne incognita and other tropical RKN species are controlled using resistant cultivars carrying Mi gene, but recent reports have shown that virulent isolates of M. incognita are capable to overcome resistance. To determine the host range of 8 M. enterolobii populations and 2 virulent isolates of M. incognita, a study was conducted within the framework of ANR (FR)/DFG (DE) project AEGONE (431627824). 20 crops species previously classified as non-, minor- or major-hosts to M. enterolobii were challenged under greenhouse conditions with 8 M. enterolobii populations (collected from different geographic areas and hosts) and 2 virulent isolates of M. incognita reared as single egg mass lines. Based on the determined reproduction factor (RF), crops were categorized as good-host (RF is ≥1), poor-host (RF between 0 and 1) or non-host (RF=0). Among 20 plant species, 10 showed RF values ≥ 1 for all 8 M. enterolobii populations and 12 showed RF values ≥ 1 for both virulent isolates of M. incognita. Although several reports suggested roses as minor-host to M. enterolobii, no reproduction was observed in all M. enterolobii populations and as well in M. incognita isolates tested. Contrary to recent reports, phacelia and fodder radish were good hosts for 5 and 7 populations of M. enterolobii, respectively. Based on the results it was demonstrated that several populations of M. enterolobii and virulent isolates of M. incognita are capable of reproducing on crops previously reported as non-hosts. Therefore, further studies are underway to investigate the potential to adapt to initially poor hosts, the related costs of fitness and to determine variations on the genome level with differences in host compatibilities. [ABSTRACT FROM AUTHOR]

Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential inttoduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platfonns revealed an analytical sensitivity of one juvenile in a background of 1,(XX) nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better perfomiance concerning detection of M. enterolobii target DNA in complex DNA backgrounds. [ABSTRACT FROM AUTHOR]

Morphological identification of spiral nematodes of the genus Helicotylenchus is a difficult task because most characters used for their diagnosis vary within species. In this paper we provide morphological and molecular characterisations of several spiral nematodes, II. broadbalkiensis, H. digonicus, H. dihystera, H. microlobus, H. paxilli and H. pseudorobustus, collected in different geographical areas of USA, Switzerland, Italy, New Zealand, Spain, UK, South Korea and Russia. We suggest that H. microlobus and H. pseudorobustus are valid species separated from each other morphologically and molecularly. Seven species with distinct molecular characteristics are also distinguished, but are not ascribed morphologically to any specific taxon because of the low number of specimens available. Phylogenetic relationships of H. pseudorobustus with other Helicotylenchus species are given as inferred from the analyses of 154 sequences of the D2-D3 of 28S rRNA gene and 37 sequences of ITS rRNA gene. [ABSTRACT FROM AUTHOR]

The article offers information on the research study done to examine the host range and damage potential of root-knot nematodes in Europe. Topics include need of Polymerase Chain Reaction (PCR) amplification for species identification, enabling of fast and reliable molecular diagnostics producing species-specific amplicons, and rearing of root nematode species is done on tomato in a glasshouse.

In the detection of plant pests, speed and accuracy are vital. High-resolution melting curve (HRMC) analysis was therefore evaluated as a new tool for the identification of root-knot nematodes ( Meloidogyne spp.). On the basis of the second intergenic spacer (IGS2) region of the ribosomal DNA cistron, Meloidogyne chitwoodi, M. fallax and M. hapla were successfully distinguished from each other and the group of the three tropical species, M. incognita, M. arenaria and M. javanica. Conversely, it was shown that the IGS2 region is not suitable for the tropical species M. enterolobii (senior synonym of M. mayaguensis) as the amplification of multiple fragments of different lengths prevented a reliable HRMC analysis. However, the obtained results provide a proof of principle that HRMC analysis can be a suitable single-tube assay for fast and accurate root-knot nematode identification. [ABSTRACT FROM AUTHOR]

The objectives of this study were to determine the efficacy, range of activity and effective concentrations (EC50 and EC80) of abamectin as a seed treatment of tomato against Meloidogyne incognita, M. arenaria and M. javanica. The study revealed that abamectin seed treatment at concentrations ranging between 0.3 and 1 mg a.s. seed-1 is highly effective against the three species of root-knot nematodes, retaining its efficacy in the soil for 8 weeks. The highest EC50 found was reached at 0.2 mg a.s. seed-1. The highest EC80 for the number of egg masses per g root in the three Meloidogyne species was attained at 0.51 mg a.s. seed-1. Abamectin as a seed treatment is promising for the control of root-knot nematodes in the field since only low amounts of active ingredient are required to give adequate protection in the most sensitive stages of tomato root growth and development. [ABSTRACT FROM AUTHOR]

Endophytic bacteria were isolated from coffee roots in Ethiopia and identified by Fatty Acid Methyl Ester-Gas Chromatography (FAME-GC). A total of 201 and 114 endophytic bacteria were isolated and identified during the wet and dry seasons, respectively. The most abundant genera were Pseudomonas, Bacillus, Agrobacterium, Stenotrophomonas and Enterobacter. Population densities were higher during the wet season than the dry season ranging from 5.2 × 103 to 2.07 × 106 cfu (g fresh root weight)–1. Culture filtrates of the bacterial isolates showed nematicidal effects of between 38 and 98%. The most active strains were Agrobacterium radiobacter, Bacillus pumilus, B. brevis, B. megaterium, B. mycoides, B. licheniformis, Chryseobacterium balustinum, Cedecea davisae, Cytophaga johnsonae, Lactobacillus paracasei, Micrococcus luteus, M. halobius, Pseudomonas syringae and Stenotrophomonas maltophilia. Bacillus pumilus and B. mycoides were most effective in reducing the number of galls and egg masses caused by M. incognita by 33 and 39%, respectively. [ABSTRACT FROM AUTHOR]

The persistence of the nematophagous fungus Paecilomyces lilacinus Samson strain 251 (PL251) and the effect of application rate, substrate type, as well as the presence of the nematode host on its dynamics after application to the soil were investigated under controlled conditions. In all experiments, increase of P. lilacinus colony forming units after application was not found. In contrast, a gradual decline in fungal densities over time was observed. Application rate had no significant effect on the dynamics of the fungal population. Likewise, P. lilacinus density decline in soil was not significantly affected by the presence of the nematode host. Substrate type had a significant effect on P. lilacinus persistence in soil. The fungal agent persisted longer in silty loam and clay soil, with reduced persistence when sand was added to field soil. Conversely, when organic substrate was added to pure sand, persistence was significantly increased. Although persistence of fungal biocontrol agents in soil depends on various biotic and abiotic conditions, baseline data on persistence such as those reported in this study are helpful for biocontrol and environmental risk assessment and merit further study. [ABSTRACT FROM AUTHOR]

Antagonistic bacteria have been repeatedly shown to be promising microorganisms for the biological control of sedentary and migratory endoparasitic nematodes. Depending on the bacteria involved, the mechanisms of action include: obligate parasitism, reduction in penetration, growth inhibition due to competition for nutrients and antibiosis associated with bioactive metabolites. In the present studies, the mode of action of the antagonist bacteria Bacillus firmus, isolated from a bionematicide, was evaluated. Significant rates of paralysis and mortality were detected after incubation of three nematode species in low concentrations of the pure culture filtrates following removal of the bacterial cells. The same culture filtrates also significantly reduced hatching of Meloidogyne incognita. Pure bacterial cell suspensions added to sand also reduced survival of R. similis in bioassays by 41% over the controls. The mode-of-action responsible for nematode paralysis and mortality was therefore demonstrated to be closely associated with the production of bioactive compounds secondary metabolites by the bacteria. [ABSTRACT FROM AUTHOR]

The interactions of Paecilomyces lilacinus strain 251 with the arbuscular mycorrhizal fungus Glomus intraradices and their significance for the control of Meloidogyne incognita on tomato were investigated in greenhouse experiments. Application of P. lilacinus had no effect on the frequency and intensity of tomato root colonization by G. intraradices. Likewise, the decline of the nematophagous fungus densities after single application in soil was not affected by the presence of the mycorrhizal fungus. Single application of P. lilacinus, as pre-planting soil treatment, resulted in significant reduction of nematode damage. In contrast, mycorrhizal inoculation did not provide sufficient biocontrol. Combined application of the two agents did not enhance root protection compared to single treatments. Double treatment of mycorrhized seedlings with P. lilacinus, as seedling drench and pre-planting soil treatment, 4 and 1 week before transplanting, respectively, resulted in the highest reduction of the nematode damage. These results indicate the potential of the commercial P. lilacinus strain 251 and mycorrhiza for integration in nematode control strategies. [ABSTRACT FROM AUTHOR]

A bioassay was developed to evaluate sugar beet for resistance to the stem nematode Ditylenchus dipsaci. To produce large numbers of D. dipsaci for inoculation of sugar beet seedlings, a protocol for a monoxenic carrot disk culture was established. Ditylenchus dipsaci multiplication was greatest with 125 000 nematodes per carrot disk after 72 days at 20°C with an initial inoculum density of 75 nematodes. Higher inoculum densities resulted in more rapid decay of carrot tissue and lower numbers of D. dipsaci. Inoculation of 200 D. dipsaci in leaf axils of sugar beet seedlings resulted in high penetration rates. The addition of 1% carboxymethyl cellulose (CMC) as a gelling agent resulted in a significantly increased penetration rate of 96% compared to tap water with 34%. Soil infestation was not suitable, with only 4 and 11% of the nematodes penetrating the sugar beet seedlings when inoculum was applied in water and CMC, respectively. The bioassay enables a rapid screening for resistance in sugar beet to D. dipsaci. [ABSTRACT FROM AUTHOR]

Paecilomyces lilacinus and in particular the commercial strain 251 has been intensively tested for biological control of plant parasitic nematodes. Since this species has been mentioned in a number of reports concerning infection of humans, the human health risk for Paecilomyces lilacinus strain 251 was investigated. The effects of time, temperature and growth medium on radial colony growth and germination were determined. Additionally, exposure to 36°C and its effect on germ-tube extension and on survival of conidia was evaluated. Radial growth was significantly affected by temperature, growth medium and their interaction. Optimum temperatures were between 24 and 30°C, but no growth was found at 36°C. Germination rate was significantly influenced by time, medium, temperature and their interactions. The optimum temperature range for germination was between 28 and 30°C. Formulated conidia were capable of germinating at 36°C. However, studies on germ-tube extension conducted at 36°C showed a delay in development for 28-49 h and no further germ-tube extension was found after exposure for 80-95 h. Slopes of survival curves were significantly influenced by the type of conidia tested. In general, conidia did not survive exposure to 36°C for 168 h. These experiments indicate the temperature conditions where the strain is likely to be active and provide supporting data for full environmental and health risk assessments of biocontrol fungi. [ABSTRACT FROM AUTHOR]

Two glasshouse experiments with sugar beet cvs Penta and Macarena inoculated, respectively, with 0 or 1500 and 0, 500, 1000 or 1500 juveniles of Heterodera schachtii, were conducted to estimate the capability of laser-induced chlorophyll fluorescence (LIF) and pulse amplitude modulated (PAM) chlorophyll fluorescence techniques to detect H. schachtii infestation and to differentiate between infestation levels. Fluorescence and gas exchange parameters, nitrogen and chlorophyll content of sugar beet leaves were measured weekly after nematode inoculation. Sugar beet plants responded to H. schachtii infestation initially with a decrease in photosynthesis rate and later with a reduction in nitrogen uptake and chlorophyll concentration. At the early stages of nematode infestation, before visual symptoms were evident, infested sugar beet plants displayed increased fluorescence (F680, F740). Later stages of infection were accompanied by an increase in the F686/F740 ratio, ground fluorescence (Fo) and a decrease in photochemical efficiency (Fv/Fm) induced by degradation of leaf chlorophyll. Sugar beet plants infested with 500, 1000 or 1500 juveniles per 100 cm3 of soil did not differ either in their nitrogen and chlorophyll content or in photosynthesis and transpiration rate. The linear discrimination analysis based on the combination of PAM and LIF parameters resulted in 100% correct classification of control plants and high classification rates (60-100%) of the infested treatments on all the sampling dates. Whether the fluorescence technique will differentiate nematode densities under field conditions needs further study. [ABSTRACT FROM AUTHOR]

The egg-pathogenic fungus Paecilomyces lilacinus strain 251 (PL215) was investigated under different temperature regimes for biocontrol activity against the northern root-knot nematode Meloidogyne hapla. Biocontrol activity by PL251 was affected by temperature and initial nematode inoculum density. At 20/23°C (night/day) control efficacy reached 71% at a high inoculum density of 10 000 eggs and juveniles (E + J) per 500 cm3 compared to 65% control at the lower inoculum density (5000 E + J/500 cm3). By contrast, average efficacy was 5% and 46% at low and high inoculum levels, respectively, when temperatures were between 19°C and 21°C (night/day). A significant interaction between the factors inoculum density and treatment with PL251 was observed at low temperatures, when only few M. hapla galls and egg masses developed after inoculation with 5000 E + J/500 cm3. When the temperature was favourable for the fungal parasite (25°C), efficacy reached 90%. In glasshouse experiments with unfavourable temperatures for M. hapla development (25°C-33°C), tomato plants repeatedly treated with PL251 showed significantly higher fruit yield (23-102%). [ABSTRACT FROM AUTHOR]

The plant parasitic root-knot nematodes Meloidogyne spp. are a devastating threat to agriculture. The urgent need to search for alternative root-knot nematode control methods that are less environmentally toxic is a demanding challenge to secure the increasing global food demand. Therefore, we combined chemical and biological control strategies to evaluate their management potential on Meloidogyne incognita infested tomatoes. To determine the combined effect of the nematicides Velum and Purpureocillium lilacinum strain 251 formulated as wettable granulate (BioAct WG), we evaluated tomato yield parameters, gall index, soil and root nematode populations. Velum is a chemical nematicide with fluopyram as its active ingredient and BioAct is a biological nematicide based on the egg pathogenic fungus Purpureocillium lilacinum (strain 251). As a single treatment, the nematicide Velum showed better M. incognita reduction at-planting, while BioAct controlled the nematode population throughout the growing season. Greenhouse experiments in two consecutive years, showed that by combining the two nematicides, the M. incognita controlling effect was enhanced and the tomato yield increased compared to single nematicide treatments. The controlling effect of P. lilacinum was lower when M. incognita population increased, presumably based on its limiting ability to parasitize the increasing numbers of nematode eggs. To conclude, we have shown that combining chemical and biological nematicides can successfully control the root-knot nematode M. incognita and increase yields. Velum downregulated the nematode population at-planting and reinforced the biological efficacy of P. lilacinum throughout the growing season. • Root-knot nematodes were controlled by combining chemical and biological nematicides. • Tomato yield increased using the nematicides Velum and P. lilacinum strain 251. • Nematode populations at-planting were crucial for the efficacy of P. lilacinum. [ABSTRACT FROM AUTHOR]