First Detection of the Potato Cyst Nematode (Globodera rostochiensis) in a Major Potato Production Region of China

The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida are the very important quarantine nematode pests of potato [Stone 1973]. Both species cause serious potato tuber yield losses. These species are subjected to strict quarantine regulations in many countries (EPPO 2017). G. rostochiensis was detected in Sichuan and Yunnan province, China in 2022 (Jiang et al. 2022). A survey for cyst nematodes in potato fields was conducted in Guizhou Province from 2018 to 2020. A total of 200 samples, including roots and soil, were collected from 40 potato fields in Hezhang (N27 06.145, E104 39.153) and Weining (N26 50.541, E104 09.885) counties in Guizhou Province, China. The Cobb decanting and sieving method was used to isolate cysts and J2s from the soil samples (Southey 1986). The potato roots were stained with acid fusion to observe cyst development. Morphological and molecular analyses indicated that 27 (13.5%) of the samples contained G. rostochiensis. The cyst density ranged between 1-85 cysts per 100 cm3 of soil and a mean density was 15 cysts per 100 cm3 soil. The smoothly rounded cysts were in brown and golden color, and the terminal cone was absent and circumfenestrate. The key morphometrics of cysts (n=20) were 695 ± 26 (685-757) μm in length excluding neck and 690 ± 30 (668 to769) μm in width; the number of cuticular ridges between anus and vulval fenestra was 16.3 ±2.1 (14 to 18); fenestral length was 15.1 ± 2.1 μm (13.18 to 19.27); distance from anus to the edge of fenestra was 61.12 ± 8.9 (49.22 to76.27) μm; and Granek's ratio was 4.54 ± 0.8 (3.97-5.26). The key morphometrics of J2 (n = 20): 468.0 ± 20.1 (427 to - 521) μm in body length, 20.58 ± 0.7 (20.2 to 21.8) μm in stylet length, 43.9 ± 5.6 (40.3 to 53.9) μm in tail length, and 23.1 ± 1.8 (21.77 to 25.32) μm in hyaline region length. The cyst and J2 morphologies were consistent with those of G. rostochiensis (Subbotin et al. 2010, EPPO 2017). Genomic DNA was isolated from cysts (n=20). DNA extraction was performed in a volume of 20 μl containing 3 μl 10× PCR buffer, 3 μl Proteinase K (600 μg μl-1), 14 μl distilled water and a single cyst was added and ground in an ice bath as described by Ou et al. (2008). The internal transcribed spacer (ITS) regions were amplified using the universal primers: rDNA1 (5'-TTGATTACGTCCCTGCCCTTT-3') and rDNA2 (5'-TTTCACTCGCCGTTACTAAGG-3') (Fleming 1998 ), and the 28S rDNA-D2/D3 regions were amplified using the primers: D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and D3B (5´-TCGGAAGGAACCAGCTACTA-3´) (Subbotin et al. 2006). After the brackets at the beginning and end of the sequences were closed-up, the ITS rDNA sequences (GenBank Accession No. MZ042367 and MZ042368) showed 99.66% - 99.92% identity to G. rostochiensis sequences available in GenBank (FJ212166.1, GQ294513, FJ212164.1 and KJ409617.1). Sequences from the 28S region (GenBank Accession No. MZ057597 and MZ057598) were 99.23% - 99.74% similar to those of G. rostochiensis isolate from Slovakia (KJ409625.1), Italy (KJ409631.1) and United Kingdom (KJ409633.1). We used species specific primers ITS5（5'-GGAAGTAAAAGTCGTAACAAGG-3'）and PITSr3 (5'AGCGCAGACATGCCGCAA-3') to amplify the product (Bulman & Marshall 1997; EPPO 2017). A single 434bp fragment was obtained from Hezhang and Weining populations. A host test for the Hezhang and Weining populations were performed by inoculating 1,000 eggs per plant of varieties Qingshu 9, Huize 2 and Hezuo 88 grown in the pots containing 800 cm3 of sterilized soil (soil: sand ratio was 3:1), and four replications were tested in greenhouse under 16 h light, 22°C in the day and 8 h dark in the night. At 90 days post inoculation, 32.6 ± 7, 31.2 ± 8, and 29.5 ± 8 females and cysts were extracted from the infected roots and soils of the varieties Qingshu 9, Huize 2 and Hezuo 88, respectively. No females and cysts were observed on the control plants. The trial indicated that potato cultivars Qingshu 9, Huize 2 and Hezuo 88 are hosts for the Hezhang and Weining populations of Globodera rostochiensis. To the best of our knowledge, this is the first detection of potato cyst nematode Globodera rostochiensis in Guizhou Province, China.