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Comparison of Globodera egg viability by primary staining with Meldola's Blue and secondary hatch of Meldola's Blue-exposed eggs with PRD.
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Laboratory-based methods to test egg viability include staining with Meldola's Blue and/or juvenile (J2) hatching assays using potato root diffusate (PRD). These two methods have not been tested under identical conditions to directly compare their assessments of Globodera egg viability. Using two bioassay strategies, cysts from a Globodera sp. popu...
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The potato cyst nematodes (PCN), Globodera rostochiensis (Woll.) and G. pallida (Stone), are important pests of potato globally. Due to their extensive damage potential and the challenge of managing them, these nematodes are under strict regulations in many countries; however, despite these regulations, PCN continue to spread into new areas and cou...
Citations
... All the 96 well plates were covered with aluminium foil to avoid evaporation and were kept in the dark inside laboratory cabinets at 25 ± 5°C. After the designated time (24, 48 and 72 hrs) of exposure elapsed, one cyst was picked from each experimental unit in each treatment, after which the cysts were incubated in 0.1% Nile blue stain for 48 hrs (Faggian et al., 2012;Kroesf et al., 2011). ...
In-vitro assays to determine the effect of plant extracts on egg viability and mortality of J2s of potato cyst nematode were evaluated. Methanol, ethyl acetate, hexane and water were used as solvents. Eggs and J2s were exposed to plant extracts for 24, 48 and 72 hrs. Treatments were arranged in a completely randomized design with three replications. Loss of egg viability and mortality of J2s significantly increased with an increase in the time of exposure to the extracts. Hexane extracts had a significantly higher loss of egg viability. Mexican sunflower extracts had a significantly higher loss of egg viability, having 93 and 89.2% non-viable eggs/cyst in experiments 1 and 2, respectively, compared to other plant extracts. This was followed by garlic, which had 89.5 % and 86.3%, and then ginger, 86.8% and 85.9% non-viable eggs/cyst in experiments 1 and 2, respectively. Garlic, Mexican sunflower and ginger after 72 hrs of treatment exposure had significantly (P<0.05) high juvenile mortalities of 64.5%, 64.9% and 70.2%. Mexican sunflower, ginger and garlic extracts were effective in inducing loss of egg viability and mortality of J2s of PCN.
... 27 Specifics of the 96-well PRD hatching assay have been described in detail previously. 28 After one week, the number of hatched second-stage juveniles (J2) was determined. After counting, cysts were removed from wells and cut open using scalpel and forceps under a dissecting microscope and the number of unhatched eggs was then counted on an inverted microscope. ...
BACKGROUND
It is challenging to manage soilborne pathogens and plant‐parasitic nematodes using sustainable practices. Here, we evaluated a novel energy application system, Directed Energy System (DES). This system generates pulses of energy capable of impacting selected biological organisms. The oomycete Phytophthora cinnamomi, the fungus Verticillium dahliae, and the plant‐parasitic nematodes Meloidogyne hapla and Globodera ellingtonae were added to soil. Then DES‐generated energy was applied to soil and impacts on target organisms were determined.
RESULTS
DES applied at 20, 40 and 70 J cm⁻³ to P. cinnamomi and V. dahliae resulted in ≥50% and 92% reductions (respectively) of propagules per gram of soil in comparison to the untreated control. There was a significant reduction of M. hapla eggs per gram of host tomato root between the untreated control, and 2.2, 13 and 25 J cm⁻³ DES dosages applied pre‐ or post‐planting. Additionally, an 84% reduction in hatch from G. ellingtonae encysted eggs after treatment with 70 J cm⁻³ DES was observed. The dosages ranged from 40 or 80V mm⁻¹ for nematodes to 200 V mm⁻¹ for fungi.
CONCLUSION
DES‐generated energy reduced survival of the soilborne pathogens P. cinnamomi and V. dahlia, and the plant‐parasitic nematodes M. hapla and G. ellingtonae. The application of this technology to a field setting remains to be considered. Published 2020. This article is a U.S. Government work and is in the public domain in the USA. Pest Management Science published by Wiley Periodicals, Inc. on behalf of © 2020 Society of Chemical Industry.
... The current vital stain used for viability assessment of PCN, Meldola's Blue, has an incubation of 7 days for adequate staining of eggs. This is time consuming and can skew in favor of a non-viable population [35]. This method is also unsuccessful for staining aged specimens, typically producing yellow-colored specimens, and in some cases, specimens may be partially stained in either the head area or at the tail end [12]. ...
... No efficient method, except time-consuming and labor-intensive manual processing and microscopic observation, is currently available for sorting cyst populations of PCN. In addition, determination of whether a PCN population is viable (live) or nonviable (dead) is essential, considering the fact that detection of only one cyst in the soil can lead to a potential threat to crop production systems (Back et al. 2004;Kroese et al. 2011) or regulatory action. A number of procedures have been used to find out which egg individuals are alive and which are not; however, variable outcomes have been reported for some of these techniques. ...
... A number of procedures have been used to find out which egg individuals are alive and which are not; however, variable outcomes have been reported for some of these techniques. Because distinguishing between live and dead eggs by visual examination under a microscope is not always possible, several conventional techniques such as egg staining with fluorescent and nonfluorescent stains (Ogiga and Estey 1974;OEPP/EPPO 2013), egg hatching in response to host and nonhost plant root diffuses (Devine et al. 1996), and a combination of egg staining and hatching (Kroese et al. 2011) bioassays have been used to determine the viability of a cyst nematode population. Many nonfluorescent stains such as Meldola's Blue, eosin Y, and Nile Blue A (Ogiga and Estey 1974;Shepherd 1962) have been extensively used for testing the viability of Meloidogyne incognita, Heterodera glycines, G. rostochiensis, and G. pallida (Kroese et al. 2011;Meyer et al. 1988). ...
... Because distinguishing between live and dead eggs by visual examination under a microscope is not always possible, several conventional techniques such as egg staining with fluorescent and nonfluorescent stains (Ogiga and Estey 1974;OEPP/EPPO 2013), egg hatching in response to host and nonhost plant root diffuses (Devine et al. 1996), and a combination of egg staining and hatching (Kroese et al. 2011) bioassays have been used to determine the viability of a cyst nematode population. Many nonfluorescent stains such as Meldola's Blue, eosin Y, and Nile Blue A (Ogiga and Estey 1974;Shepherd 1962) have been extensively used for testing the viability of Meloidogyne incognita, Heterodera glycines, G. rostochiensis, and G. pallida (Kroese et al. 2011;Meyer et al. 1988). These staining assays provide satisfactory assessment of the viability but they are time-consuming and tedious and are subjected to enumerating errors that make them unreliable for routine usage (Back et al. 2004;Beniers et al. 2014). ...
The Complex Object Parametric Analyzer and Sorter (COPAS) is a large particle flow cytometer designed for analyzing, sorting, and dispensing objects of varying sizes. We explored the potential of using this instrument to analyze and sort various developmental stages and egg viability of Globodera pallida. Cysts were successfully examined and sorted from debris by optimizing Side Scatter and Red-Fluorescence parameters on the COPAS. We were able to separate eggs and second-stage juveniles from samples of mixed population using Extinction and Time of Flight. Separation of live and dead eggs was examined following staining eggs with SYTOX Green and application of Time of Flight and Green Peak Height. Data were compared with a commonly used viability assay by which eggs were stained with Meldola’s Blue and examined by a microscope. COPAS proved to be effective in assessing viability by detecting two separate gates; live eggs having green fluorescence peaks < 190 and dead eggs with the peaks > 190. The application of COPAS in combination with SYTOX Green detected a greater number of live eggs than the Meldola’s assay suggesting that SYTOX Green provided an overestimate of live eggs. COPAS noticeably increased the accuracy and reduced the time required for screening and analyzing nematode populations.
... Globodera ellingtonae cysts were then extracted from the entirety of the sample using a USDA cyst extractor. Percentage hatch of recovered G. ellingtonae eggs was determined following methods described by Kroese et al. (2011). Briefly, cysts containing approximately 311 eggs/ cysts were placed in 10% PRD collected from potato 'D esir ee' in individual wells of a 96-well plate for 1 wk, a period of time determined to result in most of the hatch for G. ellingtonae (Zasada et al., 2013). ...
The eradication program for the potato cyst nematode (PCN), Globodera pallida, in the Northwest of the United States revolves around the use of soil fumigation. Alternative, integrated strategies are needed to continue to battle this invasive nematode. Laboratory, greenhouse, and field experiments were conducted with G. pallida and another cyst nematode found in the United States, Globodera ellingtonae, to evaluate the efficacy of a new formulated Brassica juncea seed meal extract, as well as a traditional B. juncea seed meal, as alternate eradication strategies. This is the first report on the efficacy of B. juncea seed meal extract against plant-parasitic nematodes. Rates of B. juncea seed meal greater than 2.2 t/ha and 4.5 t/ha for G. pallida and G. ellingtonae, respectively, were required for egg hatch suppression, as determined by a potato root diffusate (PRD) bioassay. Reproduction of G. pallida on potato after exposure to B. juncea seed meal at a rate of 2.2 t/ha was also significantly reduced. In the field, 8.9 t/ha B. juncea seed meal almost eliminated egg hatch of G. ellingtonae. Rates needed for Globodera spp. suppression were greatly reduced when using the B. juncea seed meal extract. When compared side-by-side, half as much B. juncea seed meal extract, 1.1 t/ha, was required to suppress G. ellingtonae egg hatch to the same extent as B. juncea seed meal. Exposure of G. pallida to B. juncea seed meal extract at 4.5 t/ha reduced egg hatch by 90% compared with a nonamended control. The ability to reduce the amount of material being applied to soil by using an extract has the potential for integration into a G. pallida eradication program.
... Thus, the time-honored test for assessing loads of these parasites has been the measurement of their egg production by the parasites (i.e. fecal egg counts through a variety of well-described methods: McMaster, Kato-Katz, mini-Flotac and other various egg concentrating methods) [2][3][4][5]. In recent years, there has been the gradual development and validation of molecular (PCR) approaches for estimating intestinal nematode presence and load, and it is likely that this type of technique will be used more commonly in the future. ...
... It was found that G. ellingtonae reproduced well on potato and tomato (Solanum lycopersicon L.) (Skantar et al., 2011) enabling the production of large number of cysts in the greenhouse. However, when greenhouse-produced eggs of G. ellingtonae were exposed to PRD they demonstrated low hatch (5%) indicating that the eggs were in diapause (Kroese et al., 2011). The ability to break diapause in greenhouse-produced eggs was of great importance to the G. ellingtonae research program to enable subsequent experimentation. ...
... Experiment 1: Two different cohorts of cysts were used: (i) greenhouse-produced cysts reared on potato 'Modoc' during 2010 that were considered to be in diapause and which exhibited little hatch in PRD, and (ii) field-produced cysts from a 2008 potato crop, considered to have broken diapause and which hatched readily when exposed to PRD. The initial hatching dynamics of eggs from these two cohorts was previously reported (Kroese et al., 2011). Dry soil, 250 g, containing either greenhouse-or field-produced cysts was placed in 8-3 28-cm nylon bags that were tied closely. ...
... The PRD was collected from 3-wk-old potato 'Modoc' plants grown and leached at USDA-ARS, Corvallis, OR, according to Widdowson (1958). Specifics of the 96-well PRD hatching assay have been previously described in detail in Kroese et al. (2011). Cysts remained in PRD for 1 wk, a period of time determined to result in the majority of hatch for G. ellingtonae (Zasada et al., 2013), at which point, the number of hatched J2 was counted in each well. ...
Globodera spp. eggs go through a diapause, which remains dormant until favorable hatching conditions are reached. Because of the regulatory concerns with cyst nematodes, it is often only possible to rear eggs for research in the greenhouse. However, hatch is often lower for greenhouse-produced eggs than for eggs obtained from the field. The goal of this research was to determine storage conditions for Globodera ellingtonae eggs produced in the greenhouse that would increase percentage hatch. Over 3 yr, G. ellingtonae greenhouse-produced eggs were stored in different environments (-20°C, 4°C, room temperature, and the field) in either dry or moist soil. Percentage hatch after exposure to the different environments was determined in potato root diffusate. Across two experiments, field-produced eggs had higher hatch rates (65.2%) than greenhouse-produced eggs (10.4%). Temperature did not have an appreciable influence on hatch of eggs stored dry in two experiments (2.8% to 8.4% and 3.8% to 8.6%), but hatch of eggs stored in moist soil was significantly higher than in dry soil at all temperatures except -20°C (26.8% and 28.7%). However, the ability of G. ellingtonae greenhouse-, microplot-, and field-produced eggs to reproduce on potato in field microplots was not different. Although it may not be possible to produce G. ellingtonae eggs in the greenhouse that have the magnitude of hatch as those produced in the field, hatching can be greatly increased by storing eggs in moist soil at either 4°C or room temperature.
... They concluded that some eggs could not be readily classified when using the stains and the results were not always consistent. Meldola's blue is the most popular and reliable staining agent to determine egg viability (15) and it has also been used to assess the egg viability of Globodera spp. when evaluating management measures (29) and conducting surveys of regulatory significance (22). ...
... Determination of viability by a hatching assay also requires a lot of time (weeks) and is subject to many factors such as diapause and the quality of the PRD (15). Our results showed that assessing the viability of both PCN species with a hatching assay lasting 10 weeks led to fewer live eggs than the other two methods. ...
Integrated management of potato cyst nematodes (PCN; Globodera rostochiensis and G. pallida) relies heavily on the determination of cyst population densities in soil as well as the viability of the eggs inside the cysts. This study aimed to optimize a quantitative method to determine the number of viable eggs of PCN based on trehalose present in live eggs. Trehalose was extracted from cysts and from a dilution series of eggs and quantified. More trehalose was detected when cysts were crushed than when left intact. Reaction volumes were adapted to the number of eggs because small reaction volumes hampered an accurate extraction of trehalose. A maximum of 10.5 eggs/μl of reaction volume should be used to obtain a significant linear relationship between detected trehalose content and egg numbers. The sensitivity of the trehalose-based method was evaluated by determining the lowest egg detection limit and was defined as five viable eggs. The reliability of this method was tested by comparing efficacy with that of two commonly used assays, visual assessment and hatching test. The trehalose-based method gave viability results similar to those of the visual assessment, which is time consuming, requires trained personnel, and can involve some subjectivity. The hatching test identified fewer viable eggs than the other two methods. In addition, the viability of dead eggs (heated and naturally dead) was tested. No false-positive results (dead eggs declared viable) were obtained with the trehalose-based method. The robustness of the test was demonstrated by measuring the viability of eggs of PCN in different experiments repeated in time. The viability assessment method based on trehalose proved to be an objective as well as sensitive, reliable, robust, fast, and cheap technique for assessing the number of viable eggs in PCN cysts.
... However, this molecular technique does not determine whether the unhatched juveniles would be able to hatch. Juveniles inside the cysts that cannot hatch are of little agronomical relevance (Kroese et al., 2011). Hatching was taken into account by Valdes et al. (2012); however, no significant effect was observed on encysted G. rostochiensis after biofumigation with Sinapis alba. ...
The aim of this study was to assess the feasibility of controlling the potato cyst nematode Globodera pallida through biofumigation with glucosinolate-rich Brassica juncea genotypes. The main glucosinolate of B. juncea is 2-propenyl glucosinolate which is the precursor of 2-propenyl isothiocyanate. Toxicity of 2-propenyl isothiocyanate to encysted G. pallida was tested in vitro. Fifty percent reduction in hatching was found within 2 h of exposure to 0.002% 2-propenyl isothiocyanate. Based on the in vitro results, we hypothesized that biofumigation with B. juncea would reduce hatching of G. pallidain vivo and higher 2-propenyl glucosinolate levels would have a stronger effect. Plant genotype, sulfur fertilization and insect herbivory affected 2-propenyl glucosinolate concentration of B. juncea. However, increasing 2-propenyl glucosinolate concentration of B. juncea did not affect G. pallida hatching after biofumigation. The absence of a biofumigation effect was most likely due to lower concentrations of 2-propenyl isothiocyanate in vivo compared to in vitro conditions. These results show that it is difficult to reach sufficiently high levels of toxicity to reduce hatching of G. pallida under realistic conditions.
... However, this molecular technique does not determine whether the unhatched juveniles would be able to hatch. Juveniles inside the cysts that cannot hatch are of little agronomical relevance (Kroese et al., 2011). Hatching was taken into account by Valdes et al. (2012a), however, no significant effect was observed on encysted G. rostochiensis after biofumigation with Sinapis alba. ...
