Biology, pathotype, and virulence of Globodera rostochiensis populations from Kenya

Seven populations of G. rostochiensis were used in this study, six populations were from Kenya and one from Germany. Five of the Kenyan populations, ‘HAR1’, ‘HAR2’, ‘RIR’, ‘KIN1’, and KIN2, were collected from potato fields in Nyandarua County, while the sixth population (‘TGN’) was from Kiambu County. The population G. rostochiensis population ‘Ecosse’ is the official reference population for testing potato genotypes for resistance to G. rostochiensis pathotype Ro1 (EPPO, 2006). This reference population was obtained from the nematode laboratory at the Julius-kühn Institut (Braunschweig-Germany), where this study was conducted under strict quarantine conditions. The G. rostochiensis populations were multiplied twice on the susceptible potato cv. ‘Désirée’ prior to the study. Except for the cysts used in experiments testing the presence of diapause, the remaining cysts were stored at 4°C for at least six months to break the diapause before they were used in this study.

Potatoes derived from tubers and in vitro propagation were used in the study. The potato cultivars included in the study were; ‘Désirée’, ‘Laura’, ‘Albatros’, ‘Amado’, ‘Seresta’, ‘Papageno’ ‘Belana’, and ‘Ribera’ all derived from tuber. ‘Connect’, ‘Caruso’, ‘Amanda’, ‘Performer’, and ‘Rossini’ were in vitro propagated. For the second set of cultivars, in vitro plants of ‘Désirée’ served as the control. In addition, six differential potato clones; S. tuberosum cv. ‘Désirée’, S. tuberosum cv. ‘Laura’, S. kurtzianum 60.21.19, S. vernei 58.1642.4, S. vernei 62.33.3, and S. vernei 65.346.19 (Kort et al., 1977) were used to test the pathotype(s) of the Kenyan populations.

Experiments were conducted in unsterile loess soil (Müller and Rumpenhorst, 2000; Mwangi et al., 2019a). Soil amendment was done at the beginning of the experiment by mixing it with slow release fertiliser (Osmocote Exact Standard^® with 15% N, 9% P₂O₅, 12% K₂O and 2% MgO) at a rate of 1.5 g per kg of soil. The glasshouse temperature was set at 18 ± 2°C and relative humidity maintained at a range of 50 to 70% throughout the experiments. Soil and air temperatures were recorded hourly using Testo^® 175T3 (Testo Ltd, UK) temperature loggers. Supplementary light was provided during winter to ensure a minimum of 12 hr light per day.

Plants were inoculated either with full cysts packed in retrievable bags made up of a 100 µm diameter nylon mesh or with an aqueous suspension of eggs and juveniles. To prepare the inoculum, a known number of cysts were soaked in tap water overnight and then crushed to free eggs and juveniles (Seinhorst and Den-ouden, 1966). A suspension of eggs and juveniles was prepared and the number adjusted to approx. 500 eggs and J2 per ml of water. For inoculation, two, 30 mm deep holes per pot were made in the soil using a plastic rod and nematodes dispensed equally into the holes using a pipette to achieve the desired initial population density (Pi) of five eggs and J2 per ml soil.

Apart from the experiment on the study of nematode biology, all other experiments were terminated after attaining 1,000 degree-days at 6°C base temperature (DD₆). This was approx. 12 weeks after inoculation. Cysts were extracted from the entire soil volume by washing the loess through a 250 µl sieve (Mwangi et al., 2019a). The cysts together with plant debris were collected on a filter paper (Macherey-Nagel GmBh and Co. KG) and left to dry at room temperature for a period of two weeks. Cysts were then separated from the debris using the acetone extraction method (Seinhorst, 1974). In all experiments, except the one testing the diapause of G. rostochiensis, recovered cysts were counted and then crushed (Seinhorst and Den-Ouden, 1966) to estimate the final number of eggs and juveniles per treatment (Mwangi et al., 2019a). Every experiment was conducted twice under similar conditions.

The development of the G. rostochiensis population ‘HAR1’ from Kenya in the roots of the resistant potato ‘Laura’ and susceptible potato ‘Désirée’ was assessed in the glasshouse experiments and compared with the reference G. rostochiensis population ‘Ecosse’. Eye-plugs were scooped from the pre-germinated tubers of the two cultivars and planted in 192 ml pots. For each of the cultivar-population combinations, 50 potatoes were planted and immediately inoculated with 10 cysts per pot. In the first experiment ‘Ecosse’ cysts were sieved using 500 µm sieve prior to inoculation. However, due to lack of enough inoculum for ‘HAR1’ the cysts used varied in size. In the second experiment, only cysts larger than 500 µm were used for both, ‘Ecosse’ and ‘HAR1’.

The cysts were packed in small retrievable bags made up of a nylon mesh and placed next to the eye-plug. Pots were randomized in the support boxes (Mwangi et al., 2019a) and transferred to the glasshouse benches. Plants were watered as required throughout the duration of the experiment.

To assess the development of the nematodes in the roots, four pots per treatment were randomly selected at 14, 18, 23, 28, 35, 42, 49, 56, 63, and 70 days after planting. Soil adhering to the roots was collected into centrifugation tubes and second stage juveniles (J2s), males and females extracted using the centrifugation flotation method (EPPO, 2013). Roots were then washed and stained in acid fuchsin (Byrd et al., 1983). The stained roots were cut into approx. 10 mm long sections and macerated in water using an ULTRATURRAX^® homogenizer (IKA^®-Werke GmbH and Co. KG) at 1 × 10⁵ rpm for approx. 30 sec. By this, nematodes at different developmental stages were freed from the root tissues into the water to form a suspension. The developmental stages of the nematodes were counted from the entire suspension using a 15 ml counting dish under a Nikon^® SMZ1270 stereo microscope. In total, 12 weeks after planting, the experiment was stopped and the final dry cysts extracted from four pots per treatment as described above.

The presence of diapause in the six Kenyan G. rostochiensis populations was investigated using two methods. First, the populations were reproduced twice on the susceptible potato ‘Désirée’. After the first reproduction the cysts were stored to break the diapause as described above. The freshly developed cysts from the second reproduction cycle were used in this experiment without storage to break the diapause. In total, 10 fresh cysts (1st generation cysts), for each of the six populations, were packed in small retrievable nylon bags described above with five replications per treatment and buried in the pot next to the tuber of the susceptible cultivar ‘Désirée’. Pots were filled with soil, randomized in the glasshouse, and watered as required. After 12 weeks, the experiment was terminated and cysts extracted and enumerated as described above. The newly extracted cysts were then immediately used for the next inoculation cycle as described above. The experiment was repeated until four generations were completed without diapause.

In a second experiment, the G. rostochiensis population ‘HAR1’ was used to test the ability of the newly formed cysts to hatch without diapause. Hatching tests were done in potato root diffusate (PRD) and tap water was used as the control. The PRD was obtained as described by Rawsthorne and Brodie (1986). In total, 20 newly formed cysts (1st generation cysts) were placed in hatching tubes (Mwangi et al., 2019b) with four replications per treatment and hatching media (PRD or water) added. The experiment was left in the dark at room temperature for 10 weeks. Hatched juveniles were counted and hatching solution replaced once per week. At the end of the experiment, the cysts were crushed to determine the number of the unhatched eggs. The hatching tests were repeated using the 2nd and 3rd generation cysts without diapause.

Due to limited availability of planting material from differential potato clones, only the pathotype of ‘HAR1’ G. rostochiensis population was tested twice in glasshouse experiments on six differential clones (Kort et al., 1977). Pre-germinated tubers of the differential potato clones were planted into 1 L pots with five replications each. In total, 14 days after planting, each pot was inoculated with 5,000 eggs and juveniles of G. rostochiensis ‘HAR1’ to achieve a Pi of five eggs and J2 per ml soil. As control, plants were inoculated with the Ro1 reference population G. rostochiensis ‘Ecosse’. The pots were left in the glasshouse for 12 weeks and the reproduction factor assessed at the end of the experiment.

Two different experiments were done to assess the virulence of Kenyan G. rostochiensis populations. In the first experiment, the virulence of three G. rostochiensis populations (‘HAR2’, ‘KIN1’, and ‘TGN’) from Kenya and the European G. rostochiensis population ‘Ecosse’ was assessed on six in vitro derived potato cultivars. At planting, the TC plants used in the study were approx. three weeks old and 30 mm tall. Plantlets of ‘Désirée’, ‘Amanda’, ‘Performer’, ‘Caruso’ and ‘Connect’, and ‘Rossini’ were removed from the growing media and the roots washed in running water followed by planting into 192 ml pots with 10 replications each. The pots were randomized in support boxes and placed on the glasshouse bench. Due to the lack of TC plants, ‘Laura’ was tested using eye-plugs in 192 ml pots. In total, 14 days after planting, each plant was inoculated with 1,000 eggs and J2s of each of the four nematode populations to achieve an estimated Pi of five eggs and J2s per ml soil.

In the second experiment, the virulence of the ‘HAR1’ G. rostochiensis population from Kenya was tested on seven tuber derived potato cultivars. The reference G. rostochiensis population ‘Ecosse’ was also used for comparison. Tubers of the ‘Désirée’, ‘Albatros’, ‘Seresta’, ‘Papageno’ ‘Belana’, ‘Ribera’, and ‘Amado’ were planted into 1 l pots with five replications. In total, 14 days after planting, each pot was inoculated with 5,000 eggs and juveniles to achieve a Pi of five eggs and J2 per ml soil. The experiment was terminated 84 days after inoculation (DAI) and the cysts extracted. Cysts were then enumerated and crushed to estimate the final nematode population density.

The cumulative degree-days (DD) were determined by calculating the average daily temperature, minus the base temperature of 6°C (Mugniery, 1978). The reproduction factor (Rf) was calculated by dividing the final nematode density (Pf) by the initial population (Pi). The relative susceptibility (Rs) as percentage of a cultivar was determined using the formula Rs = Pf_{test cultivar}/Pf_{susceptible control} × 100, where susceptible cv. ‘Désirée’ was used as the reference cultivar. The resistance level was then rated using the standard scoring notation where Rs < 1% = 9; 1.1-3% = 8; 3.1-5% = 7; 5.1-10% = 6; 10.1-15% = 5; 15.1-25% = 4; 25.1-50% = 3; 50.1-100% = 2; >100% = 1, where 1 is the most susceptible and 9 the most resistant (EPPO, 2006). Mean numbers of nematodes and cysts recovered in each of the experiments were tested for normality and homogeneity of variance using the Shapiro test and Levene’s test, respectively. As there was no significant difference (p > 0.05) between the repeated experiments testing diapause and testing virulence with tubers, data were pooled prior to the analysis of variance. Data generated from the study on biology and diapause were analyzed using ANOVA. Means that were significantly different (p ≤ 0.05) were separated using Tukey’s HSD test. Kruskal–Wallis test was used to analyze data on virulence assessed on in vitro potato plants. Treatments that were significantly different (p ≤ 0.05) were separated using Kruskal post hoc test. Statistical analysis was done with statistical software R (version 3.6.0).

In the first experiment, G. rostochiensis J2 were found in the roots of potato for all treatments at 14 days after inoculation (DAI), at 171 DD₆. The mean number recorded did not differ (p > 0.05) among cultivars and populations (Fig. 1). A few third-stage juveniles (J3) of G. rostochiensis ‘Ecosse’ were also detected in ‘Désirée’ 14 DAI. G. rostochiensis ‘HAR1’ J3 were detected in the roots of ‘Désirée’ 18 DAI (237 DD₆). At this time, the number of G. rostochiensis ‘Ecosse’ J3 was 36.3% higher in ‘Désirée’ treatment compared to the same cultivar inoculated with G. rostochiensis ‘HAR1’. The number of G. rostochiensis J2 in the roots varied among treatments (p < 0.05) with higher numbers being recorded on ‘Désirée’ (220.0 J3 per root system) and ‘Laura’ (192.3 J3 per root system) inoculated with G. rostochiensis ‘Ecosse’ compared to G. rostochiensis ‘HAR1’. The first J3 in the roots of the resistant cultivar were detected 23 DAI (302 DD₆). Overall, the number of J3 was higher in the susceptible cultivar inoculated with G. rostochiensis ‘Ecosse’ compared to the rest of the treatments. At 23 DAI fourth-stage juveniles (J4s) were detected in all treatments except for G. rostochiensis ‘HARI’ on ‘Laura’ which appeared on 28 DAI. In total, 28 days and 367 DD₆ were required for the first males and females to emerge from ‘Désirée’. There were 62% more females in ‘Désirée’ inoculated with G. rostochiensis ‘Ecosse’ than with ‘HAR1’. From 35 DAI (463 DD₆), males were detected in the roots of ‘Laura’, for both G. rostochiensis populations, until 63 DAI (840 DD₆) when the numbers diminished. The number of females extracted from the roots increased steadily from 35 DAI (463 DD₆) with more G. rostochiensis ‘Ecosse’ females counted on the susceptible cultivar compared to G. rostochiensis ‘HAR1’ throughout the experiment. However, no females were detected in resistant ‘Laura’ for either populations (Fig. 1). The first brown cyst of G. rostochiensis ‘Ecosse’ was isolated from the soil 49 DAI (646 DD₆) and for G. rostochiensis ‘HAR1’ seven days later at 741 DD_6. The mean numbers of cysts at the end of the experiment were 918 and 705 cysts per root system for G. rostochiensis ‘Ecosse’ and ‘HAR1’, respectively.

Figure 1:

The number of developing ‘HAR1’ and ‘Ecosse’ G. rostochiensis recorded in the roots and soil in the repeat experiment were similar in all treatments. However, some developmental stages were detected earlier than in the first experiment. For instance, at 14 DAI, both J2 and J3 were detected in the roots in all treatments (Fig. 2). At this date, the cumulative degree-days were slightly higher (182 DD₆) than in the first experiment. Similarly, G. rostochiensis J4 were detected five days earlier in experiment two (18 DAI at 239 DD₆). The first G. rostochiensis ‘Ecosse’ females were detected in ‘Désirée’ on 23 DAI at 309 DD₆, this was five days earlier than in the first experiment while G. rostochiensis ‘HAR1’ females were detected five days later (28 DAI at 382 DD₆). Only males were detected in the roots of ‘Laura’ and in low numbers. The number of days required to complete the life cycle was similar to the first experiment with cumulated DD₆ of 645 and 736 DD₆ for ‘Ecosse’ and ‘HAR1’, respectively. The average number of cysts at the end of the experiment was 956 and 876 cysts per root system for G. rostochiensis ‘Ecosse’ and ‘HAR1’, respectively.

Figure 2:

The G. rostochiensis Kenyan populations tested were able to reproduce without diapause (Fig. 3). The Rf values varied significantly among generations for ‘RIR’ and ‘KIN1’, but not the other four populations. In the two former populations, reproduction of the 2nd second generation was higher (p < 0.05) than the 3rd generation. The overall reproduction in the 2nd generation ranged from Rf = 9.5 ± 2 in ‘TGN’ to Rf = 26.5 ± 3 in ‘RIR’ (Fig. 3). In the 4th generation, the lowest Rf was recorded for ‘HAR1’ (Rf = 6.4 ± 1) while ‘KIN1’ had the highest (Rf = 19.3 ± 4).

Figure 3:

When G. rostochiensis ‘HARI’ was incubated in PRD for a period of 10 weeks, up to 68% of the 1st generation cysts hatched, compared to 14% hatch in water. The hatching rate was lower in the subsequent generations with 55 and 44%, respectively (Fig. 4). Hatching in water was less than 2% in the 3rd and the 4th generation cysts.

Figure 4:

Globodera rostochiensis ‘HAR1’ and ‘Ecosse’ had a high Rf on susceptible ‘Désirée’ (Table 1). There was no cyst produced on S. tuberosum cv. ‘Laura’ and S. vernei, 65.346.19 (Rf = 0), the two clones were therefore resistant to the two populations.

The Rf value on S. vernei, 58.1642.4 was <1 for both populations. Equally, both G. rostochiensis populations had a low Rf on S. vernei, 62.33.3. Only G. rostochiensis ‘HAR1’ had a Rf of more than one (Rf = 1.1) on S. kurtzianum, 60.21.19 (Table 1).

The Rf value varied between the G. rostochiensis populations ‘Ecosse’, ‘HAR2’, ‘KIN1’, and ‘TGN’. In addition, the populations had a higher Rf in experiment 1 compared to experiment 2 (Table 2). Reproduction of the four populations on in vitro cultivar ‘Désirée’ ranged from an Rf of 14.75 to 37.27 in experiment 1 compared with Rf value of 3.53 to 19.92 in experiment 2. There was no significant difference in Rf among the populations, on ‘Désirée’, except for G. rostochiensis ‘HAR2’ in experiment 1 and G. rostochiensis ‘KIN1’ in experiment 2, which had significantly lower Rfs (p < 0.05) than other populations. All G. rostochiensis populations had significantly lower reproduction (p < 0.05) on the potato cultivars ‘Rossini’, ‘Caruso’, ‘Amanda’, and ‘Laura’ with an Rf < 1 compared to ‘Désirée’, ‘Connect’, and ‘Performer’ in both experiments (Table 2).

The G. rostochiensis population ‘HAR2’ had the lowest reproduction on ‘Connect’ in the first experiment compared to the other G. rostochiensis populations with Rf values ranging from 21.8 to 31.7 (Table 2). In the second experiment, all G. rostochiensis populations had a similar Rf value on ‘Connect’ except G. rostochiensis ‘KIN1’ that was significantly (p < 0.05) lower. On ‘Performer’, G. rostochiensis populations ‘Ecosse’, ‘HAR1’, and ‘KIN1’ had similar reproduction, while ‘TGN’ had the lowest reproduction in experiment 1. The relative reproduction of all populations on ‘Performer’ was low (Rf ≤ 1) in experiment 2 (Table 2).

Despite the variability in the reproduction of the four populations between the two experiments, the Rs values of the cultivars to the G. rostochiensis populations did not differ (Table 3). The potato cultivars, ‘Rossini’, ‘Caruso’, ‘Amanda’, and ‘Laura’ had an Rs value of less than 1% and therefore were ranked as highly resistant (score = 9) according to EPPO (2006). ‘Connect’ was ranked as highly susceptible (score = 1-2) to all populations tested (Table 3). The Rs of ‘Performer’ to the G. rostochiensis populations ranged from 5 to 17%. The cultivar had an Rs of 6 for G. rostochiensis populations ‘Ecosse’, ‘HAR1’, and ‘TGN’ and a score of 4 for ‘KIN1’ (Table 3).

Reproduction of G. rostochiensis ‘HAR1’ and ‘Ecosse’ was very low on ‘Albatros’, ‘Belana’, ‘Ribera’, ‘Amado’, ‘Seresta’, and ‘Papageno’ with an Rf < 1 and Rs of less than 1% based on experiments with tuber derived potatoes (Table 4). The six cultivars scored = 9 in resistance. Reproduction of both G. rostochiensis populations on ‘Désirée’ was high but not different (p > 0.05), although ‘HAR1’ had higher Rf of 56.6 compared to ‘Ecosse’ Rf of 51.1 (Table 4).