First Report of Soybean Cyst Nematode (Heterodera glycines) on Tobacco in Henan, Central China

Abstract

Soybean cyst nematode (SCN) (Heterodera glycines) commonly infests soybean (Glycine max), but has also been reported to infest haricot bean, mung bean, adzuki bean, some species of Lespedeza and Melilotus (3), purple deadnettle (Lamium purpureum), henbit (Lamium amplexicaule), field pennycress (Thlaspi arvense), and shepherd's-purse (Capsella bursa-pastoris) (4). During 2009 to 2011, a survey for plant parasitic nematodes on tobacco was made in Xuchang, Henan Province, central China. Thirty six percent of 50 tobacco fields showed yellowing symptoms, and females and cysts of cyst-forming nematode were observed in the yellowing tobacco roots. The cysts were characterized by a lemon shape, with posterior protuberance, ambifenestrate, bullae, and underbridge present. The key morphometrics of cysts were fenestra length (38 to 44 μm) and width (34 to 40 μm), vulval silt (41 to 50 μm), and underbridge length (73 to 99 μm), all of which were similar to SCN (1). DNA was extracted by putting a single cracked cyst collected from the tobacco root to a 0.2-ml Eppendorf tube containing 10 μl double distilled water, 8 μl 10 × PCR Buffer (Mg-), and 2 μl of proteinase K (600 μg/ml) and frozen at -70°C for 30 min, then incubated at 65°C for 1 h and at 95°C for 10 min. After centrifugation at 12,000 rpm for 2 min, the DNA suspension was used for PCR amplification. Primers TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3') were used to amplify the rDNA internal transcribed spacer (ITS) region, and a PCR fragment of 1,030 bp was obtained. The sequence (GenBank Accession No. JX561139) showed 99% similarity to H. glycines strain Hg1-Ark1 (EF611124). Duplex PCR containing the universal primers D2A (5'-ACAAGTACCGTGAGGGAAAGTTG-3'), D3B (5'-TCGGAAGGAACCAGCTACTA-3') and SCAR primers SCNFI (5'-GGACCCTGACCAAAAAGTTTCCGC-3'), SCNRI (5'-GGACCCTGACGAGTTATGGGCCCG-3'), obtained a 477-bp fragment, which is specific for SCN populations (2). Based on both morphological and molecular identification, the populations of cyst-forming nematodes on tobacco from Henan, China were confirmed as SCN. Pathogenicity tests were conducted on 30 each of 50-day-old tobacco and 5-day-old soybean plants (one plant per pot), respectively, by adding 2 ml of a suspension of 1,000 eggs and J2 of cysts collected from tobacco roots. After 35 days, 20 to 35 white females could be detected in each of the tobacco roots, and the yellowing symptom on almost all of tobacco plants was observed. Although infection on soybean plants was observed, the nematodes infected in roots was just 10 to 20 per pot, and they all stayed in the infective J2 stage. Except for one J3 until 48 dpi, no mature females could be found, and the nematode population could not reproduce on soybean tested. This suggests that the cyst nematode population from tobacco is a new pathotype of SCN. To our knowledge, this is the first report of SCN parasitized on tobacco in naturally infected fields, which is a potential threat to tobacco growth and should attract worldwide attention.