Biology, pathotype, and virulence of Globodera rostochiensis populations from Kenya Author Mwangi, James M. Chuka University, P. O. Box 109 - 60400, Chuka, Kenya. jmmaina@chuka.ac.ke Author Mwangi, Grace N. International Master of Science in Agro- and Environmental Nematology, Nematology Research Unit, Faculty of Sciences, Ghent University, K. L. Ledeganckstraat 35, 9000, Ghent, Belgium. Author Finckh, Maria R. Kassel University, Nordbahnhofstr. 1 a, 37213, Witzenhausen, Germany. Author Kiewnick, Sebastian Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Field Crops and Grassland, Messeweg 11 / 12, 38104, Braunschweig, Germany. text Journal of Nematology 2021 2021-01-01 53 1 1 13 http://dx.doi.org/10.21307/jofnem-2021-003 journal article 298622 10.21307/jofnem-2021-003 dfe913d5-9507-4728-ac25-84bf5d43f65e 2640-396X 11644581 Study of G. rostochiensis biology 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 192ml pots. For each of the cultivarpopulation combinations, 50 potatoes were planted and immediately inoculated with 10 cysts per pot. In the first experiment ‘Ecosse’ cysts were sieved using 500µm 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 500µm 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. 10mm long sections and macerated in water using an ULTRATURRAX ® homogenizer ( IKA ® -Werke GmbH and Co. KG) at 1× 105 rpm for approx. 30sec. 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 15ml 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. Investigating the presence of diapause in Kenya G. rostochiensis populations 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. Testing the pathotype of G. rostochiensis from Kenya 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 1L 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. Virulence assessment of G. rostochiensis from Kenya 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 30mm 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 192ml 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 192ml 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 1l 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.