Microscopy Method to Compare Cyst Nematode Infection of Different Plant Species

The cyst nematode, Globodera pallida, is an obligate, biotrophic pathogen of potato, causing up to 80% yield loss. In the present study, a non-destructive imaging technique was used to compare the development and behavior of G. pallida in its host Solanum tuberosum and in the non-host S. sisymbriifolium. We used microscopy-rhizosphere chambers coupled with the fluorescent stain PKH26, and compared this with destructively sampled acid fuchsin staining. No significant difference (P ≥ 0.90) in G. pallida numbers was found whether stained with PKH26 or acid fuchsin for either plant species indicating no toxic effect from the vital stain. PKH26 labelled J2s successfully located and penetrated roots of both S. tuberosum and S. sisymbriifolium. Two days after inoculation, PKH26 stained G. pallida was clearly observed migrating intercellularly through root tissues of both S. tuberosum and the non-host S. sisymbriifolium. Overall, more nematodes were observed in S. tuberosum than in S. sisymbriifolium roots. No live J2s were observed in S. sisymbriifolium roots stained with either acid fuchsin or PKH26 after 8 days. Understanding the time line of development of G. pallida in S. sisymbriifolium is important towards comprehensive understanding of plant defense responses.


Introduction
Plant parasitic nematodes cause major crop losses worldwide.They attack roots and other plant parts, reducing water and nutrient uptake, and sapping plant vigor.Cyst nematodes in the genera Globodera and Heterodera are especially difficult to control, because the eggs can persist in soil for many years inside the dead bodies of females, which are called cysts.The potato cyst nematodes (PCN) Globodera pallida (Stone) Behrens and Globodera rostochiensis (Wollenweber) Behrens are major threats to potato production worldwide.Globodera pallida was first detected in the United States in 2006 in Idaho, and is regulated by both USDA-APHIS and the Idaho State Department of Agriculture.
Phytopathogenic nematodes go through six developmental stages: egg, four juvenile stages (J1 -J4), and adult.With G. pallida, up to four life stages may be present in plant roots: J2 -J4 and adult, depending on host susceptibility.The second stage juvenile is the infective stage; it migrates within the root, where it subsequently molts and initiates a feeding site.Once a feeding site is established, the nematode continues to develop into the adult stage.Both males and females are present.Adult males exit the root, then fertilize the sedentary females.After eggs are formed, the female dies, and her body serves as a protective "cyst" at the root surface.Nematode development is arrested at some point in resistant or non-host plants.
One potential control measure for PCN is the use of so-called trap crops, which are plant species that permit infection but do not support nematode development and reproduction.Solanum sisymbriifolium Lam, a nontuber-bearing solanaceous plant, has been identified as a promising trap crop for the control of G. pallida [1].Solanum sisymbriifolium stimulates egg hatching but does not support reproduction of G. pallida [1].Lack of development of G. pallida in roots of S. sisymbriifolium was observed by Sasaki-Crawley et al. [2], but observations were made by destructive sampling and staining.Ouden [3] used polyethylene bags filled with thin layers of foamed agar to observe nematode behavior of Heterodera rostochiensis, Hoplolaimus uniformis and Pratylenchus pratensis in potatoes, carrots and wheat, respectively.Atkinson et al. [4] studied the size and shape of G. pallida and Meloidogyne incognita during development in transgenic tomato by using a morphometric analysis method.
Novel approaches are required to study in vivo dynamics of G. pallida infection processes in potato as opposed to trap crops, and also to investigate potential mechanisms of resistance in different potato cultivars.
For live observation and behavioral studies of plant parasitic nematodes, fluorescent compounds such as fluorescein diacetate (FDA) and fluorescein isothiocyanate (FITC) [5]- [7] have been used.Dinh et al. [8] combined the use of fluorescent stain PKH26 and microscopy rhizosphere chambers (micro-ROC), to study the development of the cyst nematode Heterodera schachtii, the root knot nematode Meloidogyne chitwoodi and the root lesion nematode Pratylenchus penetrans in roots of Arabidopsis thaliana.PKH26 is a yellow orange fluorescent dye (excitation max.551 nm and emission max.567 nm) with an aliphatic tail and integrates into the lipid regions of the cell membrane [9].Because of its extremely stable fluorescence, PKH26 is useful when labeled cells are to be followed for extended periods as demonstrated by the four-week retention time of PKH26 in mammalian cells [10].
Here, we describe a further elaboration and application of these techniques, to compare the course of infection of G. pallida in potato roots versus the trap crop Solanum sisymbriifolium.We applied these methods for in situ observations of G. pallida J2 infection in the non-host S. sisymbriifolium compared to its host S. tuberosum.We used micro-ROC coupled with the fluorescent stain PKH26, and compared this with destructively sampled acid fuchsin staining.

Globodera pallida Culture and Preparation of Nematode Inoculum
G. pallida was propagated on the susceptible potato cultivar "Desiree" in the greenhouse at 18˚C.The identity of G. pallida was confirmed by morphological and molecular methods [11].The plants were watered twice daily with 75 ml of water and fertilized three times per week with 20-20-20 all-purpose fertilizer.After 16 weeks of growth, cysts were recovered from soil by the Fenwick can method [12] and stored at 4˚C.
To obtain second stage hatched juveniles, 20 cysts were placed in 2.54 cm 2 nylon mesh bags (McMaster Carr, USA), and dipped in 0.5% bleach for 5 min, rinsed thoroughly with sterile distilled water).After hydration in sterile distilled water for 3 days [13], hatching was induced by placing cysts in potato root diffusate containing gentamicin (1.5 mg/ml) and nystatin (0.05 mg/ml) at 20˚C in sterile 6-well plate.After 2 weeks the hatched juveniles were collected and incubated overnight in a solution containing 100 μg/ml each of ampicillin and streptomycin (w/v) on a rocker (Standard Analog Rocker, VWR, USA) at 22˚C.The nematode suspension was fur-ther treated with benzethonium chloride (0.125%) by continuous shaking for 20 min, washed 8 times by centrifugation at 4000 rpm for 10 min, and re-suspended in sterile distilled water [14].Collected J2s (300 J2s/100μL) were suspended in a solution of PKH26 (4 × 10 −6 M, as per manufacturer's protocol, Sigma Aldrich, USA), incubated for 10 mins at 22˚C with intermittent shaking (3 times for 15 s), and washed 5 times in sterile distilled water.The stained J2s were transferred to 0.01% sterile agarose [15].Plantlets were inoculated with 100 μL solution containing 2.5 J2s/μL [8].Inoculated plants were maintained under greenhouse conditions at 18˚C ± 2˚C, 16 hours light and 8 hours dark cycle for 20 days.Six replicates of each plant species were observed every two days for 20 days under an inverted microscope (Leica, DMI 3000B, Germany).Fluorescing nematodes were detected with a rhodamine filter.

Plant Material and Micro-Rhizosphere Chambers
Potato plants (Solanum tuberosum cv."Desiree") were grown in standard tissue culture media [16].Solanum sisymbriifolium seeds were planted in plastic seed trays.One-week-old tissue culture potato plantlets and twoweek-old S. sisymbriifolium seedlings were transplanted into micro-ROC chambers (Advance Science Tools LLC, WA) and kept in the greenhouse at 18˚C ± 2˚C for 3 weeks for development of roots.Sunshine mix (Sun Gro Horticulture, MA, USA) was used for the growth medium.Plants were watered once daily with 25 ml of water and fertilized with water soluble 20-20-20 all-purpose fertilizer, three times weekly.
To determine the efficacy of staining with PKH26, a second set of S. tuberosum and S. sisymbriifolium grown in micro-ROC were inoculated with a similar method as above, but development of G. pallida was observed by using the standard acid fuchsin method [17].Three replicates of S. tuberosum and S. sisymbriifolium were destructively sampled every two days up to 20 days post infection.Roots were stained with acid fuchsin [17] and observed under a stereo-microscope (Leica Microsystems, Germany).The development of G. pallida at each life stage was recorded.

Statistical Analysis
Counts of the different development stages were subjected to analysis of variance using generalized linear mixed effects procedure of SAS ® software Proc Glimmix [18].The plant species, days post infection (2, 4, 6 and 8 days), and type of stain used were treated as fixed effects in the model.Main (individual effect of each treatment factor) and interaction effect (two way interactions of individual treatment factors) among fixed factors were tested.Data being unbalanced (due to three replicates for acid fuchsin and 6 replicates for PKH26), the Kenward-Roger approximation was used to adjust the denominator degrees of freedom, and the estimated standard errors for main and interaction effects [19].Level of significance was set for P ≤ 0.05.Post-hoc multiple pairwise comparisons of treatment group means were done with the Tukey-Kramer adjustment (Tukey's Honest Significant Difference test) to control for the Type I error rate.The packages, "ggplot2" [20] in the freeware R 3.2 [21] was used to plot the graphs.For cross-comparison of the results from the linear mixed effect model fitted to combined data, an independent standard ANOVA model on J2 counts from plant samples stained with acid fuchsin, which ignored the random effects, and a separate linear mixed effect model on J2 counts from plant samples stained with PKH26 only were also performed.The plants species and days post infection (dpi) were the only fixed effects in these models.Data analyzed spanned across 10 time points (2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 dpi) for model fitted to samples stained with acid fuchsin only.Data for J2 counts are reported.

Comparison of PKH26 versus Acid Fuchsin
Application of the fluorescent stain PKH26 coupled with micro-rhizosphere chambers allowed for in situ observations of G. pallida J2 infection in the non-host S. sisymbriifolium compared to its host S. tuberosum.Immediately after labelling with PKH26, the entire bodies of G. pallida J2s were clearly visible with fluorescence microscopy (Figure 1) and remained visible for the 20-day duration of the study.Labelled J2s successfully located and penetrated roots of both S. tuberosum and S. sisymbriifolium.No significant difference (P ≥ 0.90) in G. pallida numbers were found whether stained with PKH26 or acid fuchsin for either plant species indicating no toxic effect from the vital stain (Figure 2).Overall, more nematodes were observed in S. tuberosum than in S. sisymbriifolium roots.PKH26 combined with micro-ROC allowed in situ observation of G. pallida in its host S. tuberosum including J2 migration through root tissue (Figure 3), and molt to J3 (Figure 3).Female development was not observed with PKH26 stained nematodes, but was observed when stained with acid fuchsin.

G. pallida Development in S. sisymbriifolium Compared to Potato
At 2 DPI, PKH26 stained G. pallida were clearly observed migrating intercellularly through root tissues of both S. tuberosum and the non-host S. sisymbriifolium (Figure 3).The numbers of J2s in roots increased from day 2 through day 8 and subsequently declined, for both plant species (Figure 2).At 2 DPI, the numbers of J2s penetrating and migrating in S. sisymbriifolium was the same for S. tuberosum.However, at 4 DPI, presence of J2 in S. sisymbriifolium roots was significantly less than in S. tuberosum.The maximum number of J2s in S. tuberosum at 6 days (mean ± SE: 63 ± 3) was nearly three times higher (P < 0.0001) than the maximum observed in S. sisymbriifolium at 4 days (24 ± 2) (Figure 2).
No live J2s were observed in S. sisymbriifolium roots stained with either acid fuchsin or PKH26 after 8 days (Figure 3).At 10 DPI and after, dead and deformed PKH26 labelled nematodes were still visible in roots of S. sisymbriifolium, but not in the acid fuchsin stained roots (Figure 3 and Figure 4).In S. tuberosum, G. pallida continued to develop into third and fourth stage juveniles within 16 dpi (Figure 4).Whereas in S. sisymbriifolium, G. pallida died within the root tissue, and no further development of the nematode occurred (Figure 3 and Figure 4).Resistance in S. sisymbriifolium was expressed rapidly, and plant cell death was observed at 4 DPI (Figure 5).Solanum tuberosum infected cells did not show any morphological changes as observed under epifluorescence (Figure 5(B)) but S. sisymbriifolium infected root cells auto-fluoresced, and the nematode was dead (Figure 5(C) and Figure 5(D)).Similarly, invaded cells of S. sisymbriifolium from acid-fuchsin-stained samples, had thickened walls and were discolored (Figure 4), while this response was not observed in S. tuberosum.

Discussion
Staining with PKH26 or acid fuchsin gave virtually identical results for both plant species.Thus, use of the vital stain PKH26 apparently had no deleterious effect on either infectivity or detection of G. pallida J2s.Also, these results suggest that the stain remains incorporated in the nematode body through subsequent molts.A promising research direction will be the use of this stain to follow nematode movement and development within the root.Understanding the time course of infection and progress of resistance in the non-host S. sisymbriifolium in comparison to its host S. tuberosum will guide subsequent assays to determine the genetic response to infection.
We showed that G. pallida was able to penetrate the roots of both plant species, but, the number of nematodes entered was much reduced in S. sisymbriifolium as compared to S. tuberosum.Dead nematodes were found in the roots of S. sisymbriifolium at 4 dpi, which shows total inhibition of G. pallida development in S. also reported by Sasaki-Crawley et al. [2] [23], however, contradictory to our findings, they reported the presence of G. pallida in S. sisymbriifolium up to 5 weeks post infection.

Conclusion
Micro-ROC coupled with PKH26 was first used by Dinh et al. [8] for the live in planta observation of Pratylenchus penetrans, Heterodera schachtii and Meloidogyne chitwoodi in the roots of Arabidopsis thaliana.Our study demonstrates the efficacy of live fluorescent staining with PKH26 method to study the course of development and interaction of a cyst nematode in a susceptible and resistant plant species.PKH26 staining did not affect the infection process and was an effective tool for nematode developmental studies.The advantages of using PKH26 coupled with micro ROC to study dynamics of G. pallida allowed the visualization of dead and deformed J2s in the infected host plant cells, and to better understand the timing of the plant response.Autofluoresence of cells infected with G. pallida was observed in S. sisymbriifolium roots but not in S. tuberosum roots.Infected S. sisymbriifolium cells had thickened walls and were dark in color indicative of localized cell death.Necrosis and degeneration of syncytium have been observed in resistant soybean cultivar as early as 48 hours or after 8 -10 dpi with Heterodera glycines, depending on the source of resistance [24] [25].Localized gene expression occurring at the feeding site during its establishment can lead either to the formation of a nurse cell called a syncytium as observed in a susceptible reaction [26], or to a localized cell death resulting in death of the nematode as observed in resistant or immune responses [27].

Figure 2 .
Figure 2. Mean J2 counts (±95% confidence interval) of Globodera pallida at different dpi (days post infection) in acid fuchsin and PKH26 stained roots of Solanum tuberosum and Solanum sisymbriifolium grown in micro-ROC.Data points with different letters are significantly different.

Figure 5 .
Figure 5. (A) and (C) show PKH26 stained G. pallida in Solanum tuberosum and Solanum sisymbriifolium, respectively; (B) and (D) show the infected cells of S. tuberosum and S. sisymbriifolium, respectively under green fluorescence.Compared to S. tuberosum root cells (B) G. pallida infected S. sisymbriifolium root cells exhibited cell-wall thickening and death (D); scale bar = 50 μM.