Evaluation of Fluorescent Stains for Viability Assessment of the Potato Cyst Nematodes Globodera pallida and G . ellingtonae

Potato cyst nematodes (PCNs), Globodera pallida and Globodera rostochiensis, are quarantine pests of potato which cause significant damage to produc-tion and farm gate revenue worldwide. Accurately assessing viability of PCN eggs is important for eradication and management programs. The goal of this study was to develop a quick and reliable fluorescent staining method to evaluate viability of G. pallida and Globodera ellingtonae eggs. The staining efficiency of eight fluorescent stains was evaluated using G. pallida eggs compared with the conventional Meldola’s Blue (MB) staining method. The staining efficiency of the fluorescent stains ranged from 80.33 ± 2.99 (Sytox Green) to 100% (Acridine Orange) for non-viable eggs. Two stains were further evaluated for their efficiency in assessing viability of encysted eggs from five different greenhouse-reared G. pallida cyst sources which contained both viable and non-viable eggs. For the G. pallida cyst sources, viability ofencysted eggs were estimated to be 41.02 ± 3.81 to 62.66% ± 3.12% when stained with Acridine Orange (AO) and 79.52% ± 1.54% viability for G. ellingtonae. Both staining time and stain concentration were significant for staining efficiency of released and encysted eggs. Staining time and concentration were optimized for released eggs at 4 h at 10 µg/ml and for encysted eggs at 16 h at 25 µg/ml respectively for AO. Fluorescent stains accurately and rapidly assessed percent egg viability and were determined to be as sensitive as a seven-day incubation with the Meldola’s Blue staining method.


Introduction
Biotrophic cyst nematodes are amongst the most economically damaging pests to cropping systems worldwide [1]. The potato cyst nematodes (PCNs) Globodera pallida (Stone) Behrens and Globodera rostochiensis (Wollenweber) Behrens have been estimated to cause 9% potato yield loss in the UK [2] [3]. Both G. pallida and G. rostochiensis originated in South America and spreaded to nearly all major potato growing regions of the world. Because of the ease of spread, and economic importance, potato cyst nematodes are listed as quarantine pests and subjected to strict regulation in many countries [4] [5].
In the United States, G. pallida was discovered in Idaho in 2006 [6] and is a regulated pest by the United States Department of Agriculture-Animal and Plant Health Inspection Services (USDA-APHIS) and by the Idaho State Department of Agriculture (ISDA) [7] [8]. A current map of the infestation in Idaho is available through the USDA-APHIS website: https://www.aphis.usda.gov/aphis/ourfocus/planthealth/plant-pest-and-diseaseprograms/pests-and-diseases/nematode/pcn). Phytosanitary regulations restrict potato cultivation or any other host crop in an infested field [7]. It takes only two cysts from two different soil samples with one of the cysts containing viable PCN eggs for the field to be considered infested. Infested fields are eligible to have some restrictions lifted when no viable eggs are no longer detected by the Meldola's Blue viability staining method and potato bioassays detect no reproduction of PCN [7]. One challenge for G. pallida control is the ability of encysted eggs to remain viable in soil for long periods (over 20 years) of time, and their capacity to infect potato after a long quiescent stage [9]- [11] thus making control by crop rotation difficult.
Quantifying viability of G. pallida eggs is important for both effective management, as well as eradication programs. Unfortunately, no efficient methods except time consuming and labor-intensive staining and microscopic evaluation of nematodes are available for this purpose. The conventional method of viability assessment of PCN is performed by using vital stains, such as Meldola's Blue and Nile Blue A [12]- [14]. The Meldola's Blue method is recommended by the Canada and United States guidelines on surveillance and phytosanitary actions for the G. pallida and G. rosctochiensis and also by the European Plant Protection Organization (EPPO) for routine assays of population densities from soil samples [4] [7]. But this method requires 7 days for adequately staining the eggs.
Besides the use of vital stains, in vitro egg hatching assays [15] are also used for assessing egg viability, but this method does not account for unhatched eggs that may be viable but in diapause [16].
Fluorescent dyes are seldom used for assessing nematode egg viability, but they are extensively used for precisely diagnosing cell viability and cell deaths [17]- [20]. Both cell permeant and non-permeant fluorescent dyes are available for assessing cell viability and differential staining of live and dead cells. The cell permeant dyes can freely diffuse through the membranes of live cells and the  [21]. The fluorescent dyes offer higher photo stability and brightness compared to non-fluorescent stains and do not require prolonged incubation time [22]. For nematodes, the success of staining methods depends on the stain permeability through its cuticle and lipoprotein membrane [12] [23]. The permeability is compromised by age, environmental factors such as temperature, chemical nematicides, nematode pathogens etc. [24]. In an earlier attempt, Perry and Feil [25] used the fluorescent dye, acridine orange (AO) to determine the viability of G. rostochiensis, and Twomey et al., [26] used AO as an indicator of egg permeability in G. rostochiensis caused by contact with potato root diffusate. Similarly, AO has been used for distinguishing live and dead eggs of Heterodera glycines [24] [27] and to study the relationship between juvenile hatching and fluorescence of H. glycines eggs [28]. More recently Hajihassani and Dandurand [29] assessed the fluorescent dyes Sytox Green and Sytox Live/Dead green kit staining for viability staining of G. pallida for large particle flow cytometry.
In the present study, cell impermeant fluorescent stains were assessed for PCN egg viability. The goal of this study was to develop a fast and reliable fluorescent staining method for the evaluation of PCN egg viability. The effectiveness of the fluorescent stains was compared with the classic Meldola's Blue staining method.

Potato Cyst Nematode Cysts
Two Globodera species, G. pallida and G. ellingtonae were used for the study. Five different greenhouse reared cyst sources (A-E) from Idaho population of G. pallida reared on two different susceptible potato cultivars Desirée or Russet Burbank were used. Cysts were extracted from the soil by using an elutriator 16 weeks after infestation and planting and the harvested cysts were stored at 4˚C until experimental use [30] [31]. Morphological and molecular species confirmation of the G. pallida cyst was conducted [32]. Globodera ellingtonae cysts were provided by Dr. Inga Zasada (USDA-ARS, Corvallis, Oregon).

Optimization of Stains for PCN Egg and Cyst Staining
To optimize the staining protocol 20 surface sterilized and presoaked (24 h in water) G. pallida cysts were used for each stain. The cysts were transferred to a 2 ml eppendorf tube with 50 µl Barnstead GenPure water (Barnstead GenPure UV-TOC xCAD plus, Thermo electron LRD GmbH, Stockland 3, Germany), crushed using a Tenbroeck all glass tissue homogenizer (Fisher Scientific, USA).
The egg suspension was allowed to settle for 5 minutes at room temperature and and Sytox Green stains were used. All stain dilutions were made using sterile GenPure water. For the optimization of stains, G. pallida eggs were heat killed using a modified method of Christoforo et al., [33]. The freed eggs were heat treated for 2 h at 70˚C using a heating block and subsequently cooled on an ice before staining. For each concentration, 100 µl egg suspensions with an equal volume of staining solution were pipetted into 1.5 ml eppendorf tube and incubated in the dark at room temperature. Evaluation of staining efficiency was conducted at 1, 2, 4 and 8 h incubation times. To remove the residual stain, 50 µl of the egg suspension was centrifuged at 4800 g for 3 minutes and washed with 100 µl of GenPure water. The pelleted eggs were re-suspended in 10 µl of Gen-Pure water and dispensed to a glass slidefor microscopic examination. An average of 65 eggs was counted for each replicate with three replicates per treatment, and the experiment was repeated. Fluorescent egg percentage was calculated by using the formula: % fluorescent eggs = (stained eggs/(stained + non-stained eggs)) × 100. Two stains, AO and LDS 751 from the above experiment were further evaluated for application in staining eggs from whole G. pallida and G. ellingtonae cysts. Whole heat killed (cysts were incubated in boiling water for 7 minutes and cooled on an ice bath) cysts from source A were incubated in dark at room tem-Advances in Bioscience and Biotechnology perature for 4, 8, 16 and 24 h in 10, 25 and 50 µg/ml concentration of AO and 25, 50 and 100 µg/ml final concentration of LDS 751. Thirty-six cysts for each treatment were stained and eggs from nine cysts were evaluated for each time interval at each concentration. The cysts were crushed, washed to remove excess stain and released eggs were re-suspended in 50 µl of GenPure water. Three-10 µl aliquots containing about 140 eggs per aliquot for each treatment were dispensed into a glass slide, visualized and scored under the microscope. The experiment was repeated.

PCN Viability Assessment Using Fluorescent Stains
Five different G. pallida and one G. ellingtonae cyst source was tested using AO and LDS 751 for viability assessment using the optimized staining parameters. Eggs that remained encysted from cyst sources were fluorescently stained and compared with Meldola's Blue staining assay [13]. Surface sterilized, presoaked cysts from each source were replicated 5 times for each treatment, and the experiment was repeated. After staining, cysts from each replicate were crushed, washed to remove excess stain, re-suspended in 50 ul of GenPure water, and a 10 µl aliquot containing an average of 158 eggs were dispensed and counted. Heat killed cysts from each cyst source were used as the positive control. Viability by Meldola's Blue stain was assessed after 7-day incubation time. Percent egg viability was calculated using the formula: % viability of eggs = (Non-stained Eggs/(Stained eggs + Non-stained eggs)) × 100.

Statistical Analysis
To evaluate the staining efficiency of the vital fluorescent stains a randomized complete design (RCD) of 9 stains, 4 different optimization times at 4 different concentrations of the stains were analyzed with the generalized linear mixed model (Proc GLIMMIX) procedure of SAS statistical package [34] assuming a binomial distribution for the number of stained eggs and a logit link function. The different stains, stain concentrations and staining times were treated as fixed effects and the significance of main (percent staining) and interaction effect (interaction of stain, concentration, stain* concentration) over time were tested. The level of significance was set for p ≤ 0.05.

Staining Efficiency of Heat Killed Eggs
All fluorescent stains tested indicated a non-viable egg population of 80% or more, whereas Meldola's Blue indicated an average 51.5% non-viable population. Non-viable eggs intensely fluoresced green when stained with Sytox green or AO ( Figure 1); red when stained with LDS 751; or orange when stained with the SYTO Orange series. Empty eggs were easily distinguished from intact eggs as the eggshell membrane of an empty egg was stained leaving a hollow transparent eggshell which was easily distinguished under the microscope (Figure 1). In our study, non-viable eggs were clearly distinguished from live as the dead eggs Advances in Bioscience and Biotechnology   Figure 2 Table S1 and Table S2.

Staining Efficiency of Heat Killed Cysts
Efficiency of two stains, AO and LDS 751 was further evaluated to estimate the   percentage of non-viable, heat killed G. pallida encysted eggs. No difference was found in the efficiency of these two stains whether eggs were released or remained encysted prior to staining. However, a significantly greater percentage of eggs were stained when AO was used compared to LDS 751 or Meldola's Blue.
Unlike free eggs, whole cysts required higher concentrations of both AO and LDS 751 (25 µg/ml, and 50 µg/ml respectively). Only 93.7% of the non-viable, heat killed eggs were stained at 10 µg/ml concentration in 16 h but increased to 100% when a 25 µg/ml AO concentration was used. The percent non-viable eggs stained with LDS 751 was 86.1% when using a concentration of 25 µg/ml while increasing the concentration to 50 µg/ml resulted in a 95.9% staining efficiency (Table 2). A significant interaction between staining time, concentration and stain*concentration *time was found (p ≤ 0.0001) (supplementary Table S3 and   Table S4).    Table 3).

Comparison of Fluorescent Stains vs Meldola's Blue for Assessing PCN Egg Viability
The interaction between cyst sources and stain was not significant (p ≥ 0. 15) which indicates that the cyst sources responded similarly to each viability assessment method. However, the type of stain used had a significant effect on the percent viability assessment (p ≤ 0.001) ( Table 4).

Discussion
Accurate determination of PCN egg viability is important in estimating the potential crop loss due to a nematode infestation and for appropriate regulatory and/or management actions. Even though several staining methods are in use for the detection of PCN egg viability, use of a vital stain and its visual observation is considered to be the most accurate, reliable and widely accepted method for viability assessment [33]. 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]. The eight fluorescent stains examined in this study indicate that the use of fluorescent stains is reliable for PCN egg viability assessment. Advances in Bioscience and Biotechnology Acridine Orange is a powerful stain to measure cells undergoing apoptosis and useful in fixed tissues and cells [36]. Twomey et al. [26] used the fluorescent stain AO as an indicator of egg permeability triggered by potato root diffusate in a closely related PCN species G. rostochiensis. In the present study, almost 100% of the heat killed G. pallida and G. ellingtonae eggs were stained by AO which is in agreement with Twomey et al. [26]. Staining with AO accurately estimated non-viable eggs for both G. pallida and G. ellingtonae. Meyer et al. [24] reported that AO is useful for distinguishing between viable and non-viable Heterodera glycines eggs. LDS 751 stained over 98% of the heat killed eggs within 4 h. LDS 751 undergoes fluorescence enhancement upon binding to nucleic acids but with negligible fluorescent enhancement upon binding to proteins used to discriminate intact and damaged cells. The non-viable eggs with compromised membrane allowed the dye to effectively stain the eggs a deep read color with minimal egg membrane staining. In addition, LDS 751 is used for the differentiation of apoptotic from non-apoptotic cells [37]. This property of the LDS 751 stain can be further exploited for staining an aging population of viable G. pallida eggs.
Since both AO and LDS 751 are fixable nucleic acid stains, these stains will be more stable with little or no photo bleaching over time.
The SYTO family of fluorescent stains exhibits 450 to 1000 fold fluorescence enhancement upon binding nucleic acids [22] and are extensively used to determine viability/apoptosis of various cell lines [37]- [39]. The comparatively high staining efficiency of the SYTO stains can be exploited for viability assessment of PCN under laboratory conditions. The low cytotoxic effect [39] of the SYTO stains may be an advantage for using in downstream processing, such as hatching assays. However, one disadvantages of the SYTO stains is their sensitivity to storage conditions compared to the other fluorescent stains. In a previous study, Hajihassani and Dandurand [29] used Sytox Green stain for fluorescent staining and sorting of G. pallida using flow cytometer observed an increased viability of eggs upon increasing the incubation time. The study also reported a 7% -14.6% greater viability estimate of G. pallida eggs than Meldola's Blue staining. A loss of fluorescence for the SYTO stains subsequent to the initiation of apoptotic cascade is well documented [40] [41]. Self-quenching of SYTO molecules during cell death can result in loss of fluorescence and an overestimation of the viability. Apoptotic chromatic condensation, decrease in the number of SYTO binding sites as the chromatin condensation and/or RNA degradation during apoptosis, alterations in binding of SYTO to mitochondrial DNA and decrease in mitochondrial uptake of SYTO molecules, the loss of plasma membrane integrity during the celldeath can all be possible reasons for the fluorescence loss by SYTO stains [38]- [44].
Results from this study clearly demonstrate that fluorescent stains can reliably assess viability of both G. pallida and G. ellingtonae. The fluorescent stains assessed required minimal staining time compared to the Meldola's Blue staining. Use of fluorescent stains can be further developed for fluorescence-based high throughput sorting of PCN life stages such as assessing viability of field derived encysted eggs through use of automated cell imaging systems and/or large particle flow cytometers.