Reproduction and life history traits of a resistance breaking Globodera pallida population

Globodera pallida Oberlangen and G. pallida Chavornay were used in the study. The origin of Oberlangen are potato fields in Emsland region of Lower Saxony, Germany (Niere et al., 2014) while Chavornay, which is the official reference population used in testing potato cultivars for resistance against G. pallida Pa3 (EPPO, 2006), was obtained from the JKI-Braunschweig-Germany. The two populations were maintained on susceptible cv. ‘Desiree’ in JKI-Braunschweig. Prior to the study, the populations were reproduced and subsequently stored at 4°C for a minimum period of six months to break diapause.

Potato cultivars with different levels of resistance to G. pallida Pa2/3 (JKI, 2017) were used in this study. The cultivars ‘Laura’, ‘Albatros,’ and ‘Belana’ lack resistance to G. pallida, but they are highly resistant to G. rostochiensis Ro1/4. Potato cultivar ‘Ribera’ has a partial resistance to G. pallida (score = 6), but the source of resistance could not be established. The cultivars, ‘Amado’, ‘Amanda,’ and ‘Seresta’ are rated high in their resistance to G. pallida. The three cultivars carry several resistance genes largely from Solanum vernei and S. tuberosum ssp. andigena (Hutten and Berloo, 2001; van Berloo et al., 2007). The source of resistance in ‘Euroviva’ and ‘Eurotonda’ is assumed to come from S. vernei, but this information is not publicly available. Potato cultivar ‘Desiree’ lacks resistance to PCN and therefore it was used as the reference cultivar. The potato tubers were pre-germinated in the dark at room temperature before transferring the sprouting tubers into a well-lit room for shoot hardening.

Cysts of both G. pallida populations Oberlangen and Chavornay were incubated in potato root diffusate (PRD), Zinc Chloride (ZnCl₂), or tap water (H₂O). The hatched J2s were counted weekly for a period of eight weeks. PRD was obtained from the susceptible potato cv. ‘Desiree’ grown in the glasshouse for a period of three weeks. The plants were uprooted and roots cleaned in running water to remove adhering soil and placed in a beaker with the roots suspended in 250 ml tap water. The setup was left in the dark overnight (Rawsthorne and Brodie, 1986). Thereafter, the root diffusate was filtered using filter paper (Macherey-Nagel GmBh & Co. KG). Collected PRD was diluted by adding equal volume of water and stored at 4°C for a short period prior to use, fresh PRD was continuously produced for the entire period of the experiment. A standard concentration of 3 mM of ZnCl₂ (Greet, 1974) was used alongside PRD and tap water.

Hatching assays were conducted using batches of 20 cysts from the same reproduction cycle using four replications per treatment. Hatching was done in tubes measuring 15 mm in diameter and 60 mm height with a 100 µm sieve fixed at the bottom to hold the cyst, but allow movement of hatched juveniles into the hatching media. Hatching tubes containing cysts were placed into 15 ml Falcon tubes filled with hatching medium. Cysts were soaked in tap water for a week before they were transferred into the hatching media and incubated in the dark at room temperature. The hatched juveniles collected at the bottom of the tubes were counted weekly and the hatching media renewed. Hatching was monitored over a period of eight weeks after which the cysts were crushed and the number of unhatched eggs determined.

The development of Globodera pallida Oberlangen in the roots of susceptible cv. ‘Desiree’ and resistant cv. ‘Seresta’ was studied and compared with the reference population G. pallida Chavornay. Eye-plugs were scooped from pre-sprouted tubers using a melon baller and planted in 192 ml pots containing loess soil enriched with slow release fertiliser ((Osmocote Exact Standard®) 15% N, 9% P₂O₅,12% K₂O, and 2% MgO) at a rate of 1.5 g (kg soil)⁻¹ (Müller and Rumpenhorst, 2000; Mwangi et al., 2019). In total, 120 pots containing either cv. ‘Desiree’ or cv. ‘Seresta’ were inoculated with hatched J2s of Oberlangen or Chavornay, respectively. To obtain inoculum, nematode cysts were put on a 250 µm plastic sieve placed on a funnel with a tube clamped at the bottom. The cysts were soaked in water for one week after which the water was replaced with PRD produced as described above. The setup was left in the dark at room temperature to induce hatching. After seven days, hatched juveniles were enumerated and used for inoculation. Inoculation was done 14 days after planting. In total, 60 pots with cv. ‘Desiree’ and 60 ‘Seresta’ plants were inoculated with J2s of either Oberlangen or Chavornay population. For inoculation, two-30 mm deep holes were made in the moist soil using a plastic rod and approximately 600 J2s were dispensed equally into the two holes using a pipette and the holes carefully covered with soil. Pots were completely randomized in a metal box and placed on the glasshouse bench. Plants were watered as required throughout the experiment. Glasshouse temperatures were set at 18 ± 2°C. Soil and air temperature were recorded hourly using a Testo® 175T3 (Testo Ltd, UK) temperature logger.

Seven days post inoculation (DPI), and weekly thereafter, four pots from each treatment were randomly picked and used to assess the development of nematodes in the roots. Plants were removed from the pot and the soil clinging to the roots carefully collected for the extraction of J2s and males. Roots were then rinsed in running water and the entire root system stained with acid fuchsin (Byrd et al., 1983). Stained nematodes were visually examined under the Nikon® SMZ1270 stereo microscope and nematodes at different stages of development recorded. Male nematodes were extracted from the soil using the centrifugation flotation method (EPPO, 2013).

The reproduction of G. pallida Oberlangen and Chavornay was assessed on the 10 potato cultivars listed above. For each of the cultivars, small-sized tubers were planted in 1,000 ml pots using loess soil as a substrate. Upon emergence, (approx. 2 weeks after planting) five pots each were inoculated with 5 eggs and J2s per ml soil (EPPO, 2006) of either Oberlangen or Chavornay. Inoculum was prepared by soaking cysts in water and crushing them to free eggs and J2s (Seinhorst and Den-Ouden, 1966). The eggs and J2s in the suspension were counted and adjusted to achieve an estimated number of 500 eggs and J2s ml⁻¹. The egg and J2s suspension was dispersed into four 30 mm deep holes made into the substrate to achieve the initial density (Pi) of 5 eggs and J2s per ml soil. The holes were then carefully covered with soil and the pots randomized on the glasshouse bench. The air and soil temperatures were recorded throughout the experiment as described above.

In total, 12 weeks after inoculation, the experiment was terminated and cysts extracted from each pot by washing the soil through a 250 µm bucket sieve (Mwangi et al., 2019). Cysts and plant debris retained in the sieve were collected on filter paper and the content dried at 35°C for 3 days. Cysts were then separated from the plant debris using acetone (van Bezooijen, 2006) and counted under a stereo microscope. To determine the final nematode population (Pf) and the reproduction factor (Rf), all the extracted cysts per pot were crushed and the average number of eggs per cyst estimated. All the experiments were repeated once.

The size of the cysts of G. pallida Oberlangen and Chavornay extracted from the susceptible cv. ‘Desiree’ and resistant cv. ‘Seresta’ were measured using a Nikon® SMZ18 Stereo Zoom Microscope. Measurements were taken of 40 randomly picked cysts per population and cultivar. In addition to measuring the diameter, the cyst content was also determined in batches of 10 cysts replicated 10 times, to estimate the mean number of eggs and J2s per cyst (Seinhorst and Den-Ouden, 1966; van Bezooijen, 2006).

The size of the cysts of six other virulent populations (NI-Gpa-VIR002, NI-Gpa-VIR003, NI-Gpa-VIR004, NI-Gpa-VIR011, NI-Gpa-VIR012, and NI-Gpa-VIR013) and three avirulent populations (NI-Gpa-AVI001, NI-Gpa-AVI002, and NI-Gpa-AVI003) was determined as described above. These populations had been obtained at different time period during field monitoring in the Emsland region of Lower Saxony-Germany and their virulence determined (S. Kruessel, LWK-Niedersachsen, pers. comm.). However, unlike Oberlangen, they were not reproduced in the glasshouse prior to the study.

The males of G. pallida Oberlangen and Chavornay recovered from the susceptible cv. ‘Desiree’ and resistant cv. ‘Seresta’ were measured. Male nematodes were handpicked from a water suspension and mounted on a microscopic slide with a drop of water. A cover slip was carefully placed on the drop of water containing nematodes and the slide placed briefly on a hot plate at 60°C to relax the nematode. Measurements were taken from 40 males per treatment using Nikon® SMZ18 Stereo Zoom Microscope. To measure the J2s, cysts recovered from the susceptible cv. ‘Desiree’ and resistant cv. ‘Seresta’ used in the development study above were placed in PRD to induce hatching. J2s were then handpicked and temporary slides prepared as above. Measurements were taken of 40 J2s per treatment.

The reproduction factor (Rf) of the nematodes was determined by dividing the final nematode population (Pf) by the initial population (Pi). The relative susceptibility (Rs) of the tested potato cultivars and their levels of resistance to Oberlangen and Chavornay were determined as described in the EPPO (2006) using cv. ‘Desiree’ as a susceptible reference control. Data were tested for normality using Shapiro test while Levene’s test was used to assess the homogeneity of variance. Analysis of variance was done for data on size of cysts, J2s and males as well as the mean number of eggs per cyst. Means that were significantly different (P  ≤  0.05) were separated using Tukey’s HSD test. There was no significant difference (P  > 0.05) between two experiments testing the virulence of G. pallida Oberlangen and Chavornay. Therefore, data from the two experiments were pooled prior to analysis. T-test was used to compare the mean number of cysts per cultivar between Oberlangen and Chavornay. Kruskal–Wallis test was used to compare mean number of cysts per cultivar within each of the two populations as well as comparing the cyst diameter of the virulent and avirulent populations. Means that were significantly different at P  ≤  0.05 were separated using Kruskal Post Hoc test. All statistical analyses were done using R-software version 3.6.0 (R Foundation for statistics computing) and data were plotted using SigmaPlot® 13.0.

The number of hatched juveniles of Globodera pallida Oberlangen and G. pallida Chavornay over 8 weeks was greater in PRD (P  <  0.05) than in ZnCl₂ and the control (Fig. 1). Except for the control, the percentage of hatched juveniles in PRD was significantly greater for Chavornay compared to Oberlangen between week 1 and 4 (P < 0.05). After 8 weeks, the proportion of hatched juveniles was higher in Chavornay with 67.78% compared to Oberlangen with 62.80%. Hatching in H₂O was lower than in PRD and ZnCl₂ for both populations.

Figure 1:

In both experiments, invasive juveniles of the two nematode populations were found in similar numbers at 7 DPI in the roots of the susceptible and resistant cultivars (Fig. 2). However, in the first experiment, the average number of J2s that penetrated the host within seven days was higher with 141 ± 20 J2s per root system, compared to the second experiment with 81 ± 10 J2s per root system. The number of J2s in the roots decreased significantly during the first three samplings. At 14 and 21 DPI, the number of Oberlangen and Chavornay J2s in the resistant cv. were higher (P < 0.05) than in the roots of the susceptible cv. in experiment one (Fig. 2). In the second experiment, J2s of Oberlangen were still found in roots of cv. ‘Seresta’ at 35 DPI while only few J2s were detectable in ‘Desiree’ roots. The number of Chavornay J2s molting into the progressive stages in cv. ‘Seresta’ was lower compared to Oberlangen (unpubl. data).

Figure 2:

Male nematodes were detected in the roots of ‘Desiree’ and ‘Seresta’ at 21 DPI in both experiments (Fig. 3). There was no significant difference in the number of males of the two populations recovered from the roots of the resistant cultivar ‘Seresta’. However, for ‘Desiree’, Chavornay produced more males than Oberlangen. Subsequently, the number of males recovered from the soil increased significantly reaching the climax at 35 DPI. In the first experiment, the number of males recovered on the resistant cv. between 21 and 56 DPI was significantly higher (P < 0.05) compared to the susceptible cultivar. In the second experiment, Oberlangen had a higher number of males on the resistant cultivar (P < 0.01) compared to Chavornay (Fig. 3). On the susceptible variety, the number of males remained low in both experiments. The trend remained the same until 56 DPI when males were rarely detected in the soil.

Figure 3:

The first young female nematode was recorded in plant roots at 21 DPI in both experiments (Fig. 4). The number of females of the two populations in the roots of ‘Desiree’ and ‘Seresta’ differed significantly throughout the experiment (P < 0.05). Between 21 and 42 DPI, there was an increase in the number of females on the susceptible cultivar. However, on the resistant cultivar, females were found in plants inoculated with Oberlangen, but rarely in the roots of cv. ‘Seresta’ for Chavornay. There was a higher number of Oberlangen females on cv. ‘Desiree’ compared to Chavornay in the second experiment (Fig. 4). However, the mean number of females of the two populations did not differ in both experiments. The first brown cyst was recorded at 42 DPI on the susceptible cultivar and at 49 DPI on the resistant cultivar, respectively (unpubl. data). During the first experiment, nearly all females had turned brown 56 DPI and in the second experiment one week later. There was no difference in the duration taken by the two populations to complete the life cycle although Oberlangen females exhibited a prolonged white stage compared to Chavornay.

Figure 4:

The reproduction of Globodera pallida Oberlangen and Chavornay was highest on susceptible cv. ‘Desiree’ (Table 1). The mean number of cysts on the susceptible cv. was not different between the two nematode populations (P  > 0.05). This cultivar was therefore used as the standard susceptible control and the mean number of cysts recovered from it was used in the calculation of relative susceptibility of the tested potato varieties. The number of Oberlangen cysts as well as the Chavornay cysts extracted from the tested potato cultivars varied significantly ((P < 0.01) Table 1). Except for ‘Desiree,’ ‘Albatros,’ ‘Laura,’ and ‘Belana’, Oberlangen produced significantly more cysts than Chavornay. ‘Albatros’ and ‘Laura’ potato cultivars were highly susceptible to the Oberlangen population with Rs of 69.0 and 63.3%, respectively. In contrast, the rest of the cultivars had Rs values of 50% and below, but only ‘Amanda’ was below 10%. Potato cv. ‘Albatros’ and ‘Laura’ were highly susceptible to Chavornay with Rs of 63.2 and 53.3%, respectively. In contrast, ‘Eurotonda,’ ‘Seresta,’ and ‘Amanda’ had Rs of 1% or less. Overall, the Pf of Oberlangen was higher in all the cultivars tested. In some cultivars Pf surpassed that of Chavornay over 20 fold (Table 1).

The Rf of both G. pallida populations differed significantly (P < 0.01) among the cultivars tested (Fig. 5) Oberlangen and Chavornay did not differ in their reproduction on four potato cultivars ‘Desiree,’ ‘Albatros,’ ‘Laura,’ and ‘Belana’ (Fig. 5). However, the reproduction on the remaining six cultivars differed significantly (P < 0.01) with Oberlangen leading in reproduction rates. Chavornay had an Rf  < 1 for the cultivars ‘Euroviva,’ ‘Eurotonda,’ ‘Seresta,’ and ‘Amanda’. However, the Rf of Oberlangen on these cultivars ranged between 3 and 13 which were significantly higher compared to Chavornay (Fig. 5). Overall, Oberlangen had higher Rf than Chavornay in all the cultivars tested.

Figure 5:

The cysts of Oberlangen and Chavornay obtained from the susceptible cv. ‘Desiree’ did not differ in size (P > 0.05). However, Chavornay had bigger cysts (P < 0.05) on the susceptible cv. when compared with Oberlangen cysts reproduced on the resistant cv. (Table 2). The Oberlangen cysts from the susceptible and resistant cultivar did not differ in the size (P > 0.05). There was no difference in the numbers of eggs per cyst of the two populations reared on the susceptible cultivar (P > 0.05). However, Oberlangen cysts reared on the resistant cultivar had fewer eggs (P < 0.05) compared with Chavornay on susceptible cultivar (Table 2). The number of eggs in Oberlangen cysts reared on the susceptible and resistant variety did not differ.

Furthermore, Oberlangen had significantly longer males (P < 0.01) on both, susceptible cv. ‘Desiree’ and resistant cv. ‘Seresta’ compared with Chavornay males from the resistant cultivar. Chavornay males from the susceptible cv. were slightly shorter than Oberlangen males from the same variety (Table 2). The Oberlangen and Chavornay J2s did not differ in length (P > 0.05) regardless of the variety on which the populations were multiplied on (Table 2).

Cysts of six virulent and three avirulent populations obtained from potato fields in Emsland region differed significantly (P < 0.01) in diameter. The Oberlangen cysts were of the same size as two of the six virulent populations examined (NI-Gpa-VIR004 and NI-Gpa-VIR013). Oberlangen cysts were significantly smaller (P < 0.01) than the rest of the virulent populations (NI-Gpa-VIR002, NI-Gpa-VIR003, NI-Gpa-VIR011, and NI-Gpa-VIR012), but larger than the three avirulent populations ((NI-Gpa-AVI001, NI-Gpa-AVI002, and NI-Gpa-AVI003), Fig. 6).