First Record of Aphelenchoides besseyi on Foxtail Millet (Setaria italica) in Henan Province of China

Aphelenchoides besseyi is an important parasitic nematode on rice, reducing yields by ∼10 to 20% (Jones et al. 2013). Foxtail millet (Setaria italica) has been a major cereal crop in Northern China, especially in semiarid areas, for millennia. In August of 2019 and 2020, nematodes were surveyed on autumn grain crops. In a foxtail millet field (N34° 58′ 27″, E113° 39′ 59″) in Yuanyang County, Henan Province, some upper leaves had chlorosis with or without necrotic tips, flag leaves had crinkling and distortion, stalks were colored, earheads were vertical, glumes were brown or light black and open, and grains became thin. Ten samples were collected, and nematodes were isolated from spike pieces by the shallow plate method and counted under a stereomicroscope. The average nematodes per earhead was 1,738.75 ± 107.72. Females were slender with a short stylet, an oval metacorpus with a distinct valve, a labial region slightly wider than the first body annulus, and a conoid tail with a terminus bearing a star-shaped mucro with four pointed processes. Females measured (mean ± SD; n = 20): body length (L) = 668.92 ± 12.73 µm (647.38 to 689.70 µm); maximum body width (W) = 14.35 ± 1.11 µm (12.12 to 16.88 µm); L/W = 46.83 ± 2.94 (40.44 to 50.03); tail length = 38.93 ± 3.48 µm (33.41 to 45.92 µm); L/tail length = 17.31 ± 1.44 (14.47 to 19.62); and stylet length (ST) = 11.57 ± 0.57 µm (10.77 to 12.34 µm). Males had three pairs of ventrosubmedian papillae with the first one adanal, spicula curved with a slight basal process, terminus bearing four mucrones arranged variably, and the whole worm in a ‘J’ shape. Males measured (mean ± SD, n = 20): L = 606.66 ± 10.70 µm (586.49 to 626.37 µm); W = 13.95 ± 0.60 µm (12.71 to 14.94 µm); L/W = 43.55 ± 1.69 (40.73 to 46.43); tail length = 35.54 ± 1.93 µm (31.41 to 38.18 µm); L/tail length = 17.07 ± 0.79 (16.05 to 18.67); ST = 11.53 ± 0.56 µm (10.61 to 12.76 µm). All key morphometrics were consistent with those of A. besseyi from Brazil (Favoreto et al. 2018) and China (Lin et al. 2004; Ou et al. 2014). Amplification of rDNA internal transcribed spacer (ITS) fragments generated a PCR fragment of 830 bp from a single nematode using the primer set TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′) (Joyce et al. 1994). Five independent PCR experiments were conducted, and all PCR products were purified and sequenced. A nucleotide sequence of ITS-rDNA was deposited in GenBank (OK090549.1). It was >99% identical to those of A. besseyi from China (MW216945.1) and India (JF826518.1, JF826519.1, and JF826517.1). These results support that the nematodes were A. besseyi. Species-specific primers for A. besseyi (BSF, 5′-TCGATGAAGAACGCAGTGAATT-3′ and BSR, 5′-AGATCAAAAGCCAATCGAATCAT-3′) were used for PCR confirmation (Cui et al. 2010). An expected PCR fragment of 312 bp was obtained, consistent with those of A. besseyi reported previously. Pathogenicity was confirmed by infection of foxtail millet (cv. Yugu33). Foxtail millet budding seeds were sown in pots containing 150 ml of sterile soil mixture. In 2 weeks, 10 seedlings were inoculated with 100 A. besseyi each, and 4 plants were noninoculated as controls. Seedlings were grown in a growth chamber at 25/30°C under 12 h dark/light. On average, 73.3 and 138.2 A. besseyi were isolated from each plant at 15 and 40 days post inoculation, respectively. Both morphological and molecular characteristics were identical with the original nematodes. All upper leaves of inoculated plants had chlorosis and necrosis, symptoms similar to those observed in the field. Neither symptom developed on control plants, nor were nematodes reisolated from control plants. To our knowledge, this is the first record of A. besseyi on foxtail millet in Henan, an important grain-producing area in China. A. besseyi is an important domestic quarantine nematode, and may become a severe threat to cereal production in Henan. Our findings will be very beneficial for A. besseyi management and further research on foxtail millet in Henan.