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).

Eight cell impermeant fluorescent stains, Acridine Orange (AO), Laser Dye Styryl 751 (LDS 751), SYTO Orange 81, SYTO Orange 82, SYTO Orange 83, SYTO Orange 84, SYTO Orange 85 and Sytox Green were used to assess the PCN egg viability. All fluorescent stains were purchased from Thermo Fisher Scientific, Eugene, Oregon, USA. The vital stain Meldola’s Blue (8-dimethylamino-2,3-benzophenoxazine hemi (zinc chloride) salt) used for the evaluation of fluorescent staining method was procured from Spectrum Chemical, New Brunswick, NJ, USA.

Examination of stained eggs was conducted using a Leica DMi8 fluorescent microscope (Leica microsystems CMS GmbH, Wetzlar, Germany) equipped with a metal halide light (Lumen 200 Fluorescence Illumination Systems, Prior Scientific Inc., Rockland, MA, USA) source. A manual filter turret with filter cubes, 4’,6-Diamidino-2-Phenylindole, dihydrochloride (DAPI) (Ex:390/70-DC:455-EM:470), fluorescein isothiocyanate (FITC) (Ex:470/40-DC:495-EM:525/50), Rhodamine (Ex:546/10-DC:560-EM:585/40) and Y5 (Ex:620/60-DC:660-EM:700/76) were used for visualizing different stains with varying fluorescent emission optima. Meldola’s Blue stained eggs were analyzed under bright field. Fluorescence data for each stain were calculated and the fluorescent microscope is calibrated with focal check fluorescence microscope test slide #3 (Thermo Fisher Scientific). Photographs were taken in a darkened room at either 5×, 10× or 20× magnification with a Leica DFC 450 digital camera and processed with Leica Application Suite (LAS) software.

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 then centrifuged at 4800 g for 3 minutes at 4˚C using a refrigerated tabletop centrifuge (Eppendorf centrifuge 5810R, Eppendorf AG, 22331, Hamburg, Germany). The supernatant was removed and eggs resuspended in 1.5 ml GenPure water. Four different concentrations of stains, 5, 10, 25 and 50 µg/ml, for AO and LDS 751; and 5, 10, 25 and 50 µM/ml concentration of SYTO Orange 81 - 85 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 GenPure 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 temperature 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.

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.

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.

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

Figure 1. Acridine orange stained G. pallida eggs. The dead eggs are characterized by the deeply stained green fluorescent color whereas the live eggs with no intense fluorescence. The live eggs and empty eggs shells were distinguished by the granularity (absence of internal structures for the empty eggshell). Scale bar = 50 μM.

consistently gave a maximum fluorescent detection value of 255 whereas the live eggs have a mean value < 80 using LAS software.

All fluorescent stains required 4 h of staining and no increase in efficiency after 8 h of incubation (Table 1). Among the various stains tested, AO was the most efficient stain, as 87.61% ± 1.12% non-viable eggs were stained after 4 h at the lowest concentration tested (5 µg/ml); and 100% of the non-viable eggs were stained after 4 h at 25 µg/ml concentration (Table 1). When AO was used at higher concentrations (25 & 50 µg/ml), additional washing was required to remove high fluorescence from the background. An average of 98. 45% ± 0.86% of non-viable eggs were stained by LDS 751 (25 µg/ml) in 4 h (Table 1). SYTO Orange series 81 through 85, and Sytox Green stains (50 µM/ml) were less efficient as only 83% - 93% of the non-viable, heat-killed eggs were stained after incubation for 4 h (Table 1). Increased concentration of SYTO stains and incubation time did not increase staining efficiency (Table 1). Figure 2 shows the fluorescence of G. pallida eggs and juveniles stained with the fluorescent dyes AO, LDS 751 and SYTO Orange 85 with the control Meldola’s Blue stain. The interaction between staining time, concentration and stain*concentration *time was significant (p ≤ 0.0001). The interaction of concentration of stains and staining time on percent staining of released non-viable G. pallida egg is provided as supplementary Table S1 and Table S2.

Efficiency of two stains, AO and LDS 751 was further evaluated to estimate the

*Values are means of two independent experiments with three replicates each followed by standard errors.

*Values are means of two independent experiments with three replicates each followed by standard errors.

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).

*Values are means of two independent experiments with three replicates each followed by standard errors. GP—G. pallida, GE—G. ellingtonae.

*The data is based on 5 different G. pallida cyst sources and means with different letters are significantly different as determined by Tukey’s HSD comparison testat p < 0.05.

Viability assays using either AO or LDS 751 were compared to the Meldola’s Blue protocol. Egg viability of the five greenhouse reared G. pallida cysts sources ranged from 41.0% ± 3.8% (Source A) through 62.3% ± 3.1% (Source E) when stained with AO; 47.3% ± 1.3% through 65.2% ± 3.4% when stains with LDS 751; and 39.2% ± 6.2% to 52.6% ± 4.0% with Meldola’s Blue staining (Table 3). 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).