First detection of the potato cyst nematode, Globodera rostochiensis, infecting potato in the central region of Colombia.

Potato cyst nematodes (PCNs; Globodera spp.) cause significant losses in worldwide cultivated potato (Solanum tuberosum) crops. In Colombia, PCN was first reported in 1970 (Baeza 1972), although this report lacked a comprehensive species description and diagnosis. After that, G. pallida has been the only PCN species reported affecting potatoes in the main producing regions of Colombia (Evans et al. 1975; Nieto et al. 1983; Vallejo et al. 2021). However, in the survey conducted by Vallejo et al. (2021), a single sample from Chocontá, Cundinamarca in the central region of the country (N 5,22396046668291, W -73,6571338400244) showed molecular characters similar to G. rostochiensis. As correct identification is essential for effective pest management, the location was re-sampled in September 2022. From the soil samples collected, PCN cysts and second-stage juveniles (J2s) were retrieved from soil using Fenwick and centrifugation methods, respectively. Morphometric characters of cysts (n = 53) were consistent with G. rostochiensis, with a length without neck (L) ranging from 451 to 614 μm (X̅ = 546.9 ± 20.3 μm), width (W) from 424 to 658 μm (X̅ = 546.9 ± 25.5 μm) and L/W ratio was 1.00 ± 0.02. Distance from anus to vulva varied from 41 to 109 μm (X̅ =75.67 ± 13.8 μm), Granek's ratio from 2.3 to 5.5 μm (X̅ = 3.89 ± 0.7 μm), and the number of cuticular ridges between the vulva and the anus were 14 to 20 (X̅ = 16.19 ± 1.7). The second-stage juvenile (n = 90) length ranged from 394 to 547 μm (X̅ = 495.62 ± 31.0 μm), the stylet length varied from 18 to 24 μm (X̅ = 21.21 ± 0.9 μm) with rounded knobs. The length of the hyaline tail ranged from 20 - 31 μm (X̅ = 24.09 ± 1.92) and the true tail from 31- 56 μm (X̅ = 48.30 ± 5.71 μm). Molecular analyses confirmed morphological identification. DNA was extracted from cysts and J2s. PCR was performed for the 28S rDNA D2-D3 segment using primers D2A and D3B (Subbotin et al. 2006), and for the mitochondrial COI gene region using primers JB3 and JB5 (Derycke et al. 2005). BLAST analyses of target 28S rDNA D2-D3 sequences (OP293373-OP293380) showed 100% identity of the 658 bp to other sequences on Genbank, including isolates from Turkey, United Kingdom, and Iran (MK311329.1, MG994942.1, KU297659.1, and KU297658.1). Similarly, the target COI region sequences (OP297993-OP298001) were 100% identical to the 407 bp of G. rostochiensis POT01 isolate from Germany, and 99.75% identical to voucher NRM67 from Indonesia, and isolate CD2200 from USA (MF773722.1, MT240262.1, and MN095979.1). Phylogenetic analysis of both gene regions strongly supported G. rostochiensis, with the Colombian sequences clustering with MH399815.1, and KU297654.1 isolates for the COI and 28S regions, respectively (Fig. 1S). In addition, a pathogenicity test was conducted in the greenhouse. For this, ten cysts were inoculated to potato plants of Criolla variety grown in 5 pots of 15 cm diameter with sterile soil and sand (1:1). Noninoculated plants served as controls (three replicates each). After three months, 54 ± 23 cysts/100 g of soil were isolated from inoculated plants (Fig. 2S), resulting in a reproduction factor (R=Pf/Pi) of 4.54 ± 0.86, while no yellow females or cysts were observed on the control plants. To our knowledge, this is the first report of G. rostochiensis in Colombia. This is an important pest that causes serious yield losses of potatoes and is a quarantine nematode in many countries (EPPO 2017). Further studies are necessary to prevent the spread of this PCN species in the main producing potato regions of Colombia.