Maize (Zea mays L.) is one of the most important crops worldwide. In September 2020, five maize plants and soil samples were received from a farmer. The samples were collected from a maize field in Fugu County (N 39.02805, E 111.06723), Shaanxi Province, China. These maize plants had the symptoms of stunting or thinning. Soil nematodes were extracted from soils with modified Baermann funnel method for 48 h (Barker 1985). Trichotylenchus sp. were detected with populations ranging from 12 to 45 (in five soil samples) nematodes per 100 gram of fresh soil. Maize were planted in pots with soil samples containing Trichotylenchus sp. to propagate this plant-parasitic nematode at 20-28 °C in a greenhouse. After plants had grown for 60 days, active Trichotylenchus sp. were observed and picked under an anatomical lens (SZX16, Olympus, Tokyo, Japan) for an inoculation experiment. Freshly germinated maize (Limin 33) seeds were planted into paper pots with a sterilized mixture of soil and sand (4:1). Four plants were inoculated with approximately 2800 mixed-stage nematodes. The maize plants were allowed to grow under room conditions (15-20 °C) for two weeks and transferred to plastic pots (12 cm diameter × 10 cm deep) with the same soil mixture. After 30 days, all inoculated plants displayed the disease symptoms such as hypoplasia of fibrous roots and a hole at the base of the maize stem, but the Trichotylenchus sp. was not detected in maize roots and stems. The control plants did not exhibit any disease symptoms. The nematodes from the inoculated pots were identified by both morphological and molecular methods. The bodies of female and male were usually C-shaped when killed by hot water. The stylet was well developed with basal knobs. The posterior part of esophagus basal bulb did not overlap dorsally with the intestine. The female had a subcylindrical tail, but the male had a conical tail. The spicule of male was ventrally curved. The gubernaculum was well developed. Morphometric data based on females and males were presented as means (range). For female (n= 20): L = 1142.7 (1002.3 to 1313.4) μm, St = 26.5 (23.9 to 29.4) μm, a = 32.8 (27.4 to 38.7), b = 6.8 (6.0 to 7.9), c = 16.3 (14.9 to 19.4), c' = 2.7 (2.3 to 3.1), v = 51.3 (30.3 to 54.8), T = 70.1 (59.0 to 78.9) μm. For male (n= 20): L = 1093.3 (960.6 to 1183.7) μm, St = 24.3 (22.1 to 26.1) μm, a = 34.9 (31.1 to 39.1), b = 6.5 (5.6 to 7.4), c = 15.1 (13.6 to 16.7), c' = 3.6 (2.9 to 4.3), T = 72.5 (63.8 to 81.1) μm, SL = 28.8 (22.2 to 32.5) μm, GL = 11.5 (7.2 to 16.1) μm. The characteristics were coincident with the description of Trichotylenchus changlingensis (Guo et a., 2015). Genomic DNA was extracted from 10 nematodes, and PCR amplifications of the D2/D3 region of 28S rRNA were performed using universal primers D2A/D3B (Castillo et al. 2003), and that of the internal transcribed spacer (ITS)-rRNA region were amplified using universal primers TW81/AB28 (Amiri et al. 2002). A 780 bp amplicon of the 28S rRNA (GenBank accession no. OM276857.1) had 90.6% (Query Cover = 95%) identity with that of Tylenchorhynchus mediterraneus (KJ461557.1). Three ITS-rRNA region sequences (GenBank accession nos. OM294652.1, OM294653.1 and OM294654.1) were obtained, and the comparison revealed that sequences OM294652.1 and OM294653.1 had 98.90% (Query Cover = 100%) identity with the ITS-rRNA region sequence of T. changlingensis isolate (MH545694.1) from China, and OM294654.1 showed a 99.09 % (Query Cover = 99%) similarity to T. changlingensis sequence (MH545693.1). The morphological and molecular characterizations confirmed that the observed nematode was T. changlingensis, which is distributed in Heilongjiang, Jilin, Liaoning, Hebei, Gansu provinces and Inner Mongolia of China (Guo et al. 2020). To our knowledge, this is the first report of T. changlingensis infecting maize and causing disease in Shaanxi Province.
Keywords: Maize; Trichotylenchus changlingensis; molecular identification; morphological identification.