First Report of the Cyst Nematode <i>Heterodera koreana</i> Parasitizing Bamboo (<i>Phyllostachys reticulata</i>) in Georgia, U.S.A.

Soil and root samples were collected from two plots (5.5 x 6 m) cultivated with bamboo Phyllostachys reticulata (syn. P. bambusoides) at the USDA Southeastern Fruit and Tree Nut Research Station in Byron, Georgia, USA, in November 2021, December 2022, and December 2023. Each soil sample consisted of five randomly collected subsamples, which were analyzed for the presence of plant-parasitic nematodes as part of regular monitoring. No aboveground symptoms were seen in the field plots, but roots showed discoloration and stunted growth (Fig. 1A). An average population density of 160, 36, and 188 second-stage juveniles (J2) of cyst nematodes (Heterodera spp.) per 100 cm3 of soil was detected in 2021, 2022, and 2023, respectively. To confirm the cyst nematode parasitism, three healthy P. reticulata plants were transplanted into 10 cm diameter pots filled with nematode-infested soil and grown in a greenhouse at 25-28°C. After three months, young cysts containing eggs protruding from the bamboo roots were seen, confirming nematode parasitism (Fig. 1B and C). Morphological and molecular analyses were conducted to identify the nematode species. The J2 body shape was cylindrical with three incisures in lateral field and well-developed rounded stylet knobs. Measurements of J2 (n = 10) included the length (range = 406.0-518.1 μm, mean = 458.2 ± 16.1 μm) and width (16.0-17.2 μm, 16.6 ± 0.5 μm) of body, labial region height (3.0-3.6 μm, 3.2 ± 0.2 μm), stylet (17.2-18.9 μm, 18.02 ± 0.6 μm), tail (61.1-65.9 μm, 63.1 ± 5.8 μm), body width at anus (9.7-12.4 μm, 10.8 ± 0.3 μm), and hyaline tail terminus (35.4-53.0 μm, 44.0 ± 3.8 μm). The cysts (n = 5) were relatively lemon-shaped, light to dark brown in color with projected neck and vulval cone that lacked fenestration, bullae, and underbridge. Morphometrics were body length including neck (420.5-745.0 μm, 550.4 ± 72 μm), body width (345.0-544.1 μm, 430.1 ± 59 μm), and L/W ratio (1.0-1.4 μm, 1.1 ± 0.1 μm). The morphometric of vulval cone included fenestral length (48 to 69 µm, 60.2 ± 4.0 µm), fenestral width (43 to 51 µm, 48.9 ± 4.0 µm) and vulval slit (47 to 58 µm, 49.1 ± 2.6 µm). Morphology and morphometric measurements of the cysts and J2 (Fig. 2A-C) were comparable to previous reports of Korean cyst nematode, H. koreana from other Asian countries (Mundo-Ocampo et al., 2008; Wang et al., 2012; Tanha Maafi and Taheri, 2015; Sekimoto et al., 2017). For molecular diagnosis, DNA was extracted from single cysts (n= 3) using DNeasy Blood and Tissue Kit, and 28S rRNA and partial cytochrome oxidase I (COI) gene fragments were amplified using D2A/D3B and Het-coxiF/Het-coxiR primer sets, respectively according to Subbotin et al. (2015) and Hajihassani et al. (2018). PCR products were then purified by a DNA Clean & Concentrator kit (Zymo Research, CA, USA) and sequenced at Genewiz (South Plainfield, NJ, USA). The resulting sequences were deposited into the GenBank database and subjected to BLASTn searches. Sequence analysis of the 28S rRNA gene (GenBank accession number OL812726) showed that the H. koreana isolate from Georgia was 100% identical (692/692 bp) with H. koreana populations from Japan and South Korea. Analysis of the H. koreana isolate from Georgia based on the COI sequence (OL813218 and OL813219) revealed 100% similarity (421/422 bp) with a population of H. koreana from Japan and 98% similarity (409/409 bp) with a population from South Korea. To our knowledge, this is the first report of H. koreana parasitizing P. reticulata in Georgia as the second state in the USA. This nematode was first reported in Florida infecting fish pole bamboo (P. aurea) propagated from plant material from the Far East (Inserra et al. 1999). This report expands the information on the geographical distribution of H. koreana. Given the significant potential for damage to bamboo, precautions should be taken to prevent the nematode's spread to unaffected regions.