Associated with Ramie Root in Yuanjiang, Hunan Province, China
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SHORT COMMUNICATIONS 583 Patel, O.V., Takenouchi, N., Takahashi, T., Hirako, M., Sasaki, Pakistan J. Zool., vol. 46(2), pp. 583-586, 2014. N. and Domeki, I., 1999. Res. Vet. Sci., 66: 129-133. Patel, O.V., Yamada, O., Kizaki, K., Todoroki, J., Takahashi, A New Record of Paratylenchus T., Imai, K., Schuler, L.A. and Hashizume, K., 2004. Mol. Reprod. Develop., 69:146-152. lepidus (Nematoda: Tylenchulidae) Schnler, L.A., Shimomura, K., Kessler, M.A., Zieler, C.G. and Associated with Ramie Root in Bremel, R.D., 1988. Biochemistry, 27: 8443-8448. Singh, K., Ambler, G.R., Breier, B.H., Klempt, M. and Yuanjiang, Hunan Province, China Gluckman, P.D., 1992. Endocrinology, 130: 2758-2766. 1 1 1 Soares, M.J., Muller, H., Orwig, K.E., Peters, T.J. and Dai, G., Yongting Yu, Huiling Liu, Liangbin Zeng, 2 1 1998. Biol. Reprod., 58: 273-284. Gang Zhang and Aiguo Zhu * 1 Takahashi, T., 2006. Anim. Sci. J., 77: 10-17. Institute of Bast Fiber Crops, Chinese Academy of Takahashi, T., Hirako, M., Takahashi, H., Patel, O.V., Agricultural Science, Changsha 410205, P.R. China 2 Takenouchi, N. and Domeki, I., 1997. J. Vet. Med. Sci., College of Pharmacy, Shaanxi University of 59: 287-288. Chinese Medicine, Xianyang 712000, P.R. China Ushizawa, K. and Hashizume, K., 2006. Anim. Sci. J., 77: 18- 27. Abstract.- In this paper, for the first time, Wooding, F.B., 1982. J. Reprod. Fertil., 31(Suppl 31): 31-39. we report a new record of Paratylenchus Yamamoto, Y., Yamamoto, K., Watanabe, F. J., Stanfield, F.J. lepidus. The nematode was isolated from and Allen, W.R., 2011. Placenta, 32: 5060510 (Cherk). rhizosphere of ramie in Yuanjiang, Hunan province, China. The morphological characters Yamada, O., Todoroki, J., Kizaki, K., Takahashi, T., Imai, K., of this population are very similar to P. lepidus Patel, O.V., Schuler, L.A. and Hashizume, K., 2002. previously reported from Taiwan. Additionally, Reproduction, 124: 427-437. ribosome DNA internal transcribed spacer (ITS) sequences of this population showed 85% (Received 4 September 2013, revised 10 January 2014) identity with that of P. lepidus from Taiwan. Key words: Ramie, pine nematode, morphology, ITS. Ramie (Boehmeria nivea L. Gaud), also called “China grass”, is a herbaceous perennial plant mainly planted in China, India and other Southeast Asian and Pacific Rim countries (Liu et al., 2013). For thousands of years, ramie is well known for its excellent fiber. More recently, its leaves and shoots were found to be good feed for goose and cow. As perennial plants, the storage roots of ramie are fat and fleshy and are attractive to larvae of cockchafer (Anomala corpulenta Motschulsky, Anomala exoleta Faldermann) - popularly known as white grub, larvae of Paraglenea fortunei (Saundeas) and nematode (Yu et al., 2011; Liu et al., 2012; Zhuo et al., 2013). In order to investigate plant parasitic nematode associated roots of ramie, a survey was performed in four provinces (Hunan, Hubei, Sichuan and Jiangxi) and a municipality (Chongxing) of China, in 2010 to 2011. Pin _______________________________ * Corresponding author: [email protected] 584 SHORT COMMUNICATIONS nematodes were isolated from samples from glycerin process. Photomicrographs were taken with Yuangjiang of Hunan, Dazhou of Sichuan and a digital camera linked to a computer, and Fuling of Chongqing. Among them, abundant pin measurements were made with the aid of imaging nematodes were isolated from Yuanjiang’s samples. software (Olympus DP-soft). The nematodes were preliminary identified as Paratylenchus according to body traits, such as fat, DNA extraction and PCR application short body and long stylet (Liu et al., 2012). Pin DNA of single nematode was extracted by nematodes are known to live outside of the plant proteinase K technique as described by Yu et al. roots and only feed epidermic cells. Although the (2011) with minor modification. Single nematode pin species doesn’t result in serious yield loss of was firstly surface sterilized with 70% ethanol for 5 ramie, the wound it made would facilitate the min and washed twice with double distilled water. subsequent infection of other soil-borne pathogens, Then the single nematode was transferred into a 200 such as Rosellinia necatrix. µl PCR tube (containing 8 µl double distilled water Identification of the pin nematode was mainly and 1 µl 10×PCR Buffer). The tube was submerged based on characters of morphology and rDNA-ITS in liquid nitrogen for 3-5 min, subsequently sequence. Chen et al. (2007, 2009) identified P. incubated at 85°C for 3 min, then put in ice and 1 µl lepidus from tea plantations and P. minutus of proteinase K (2 mg/ml) was added. The mixture associated with sugar apple, sweet orange and was incubated at 56°C for 15 min and 95°C for 10 buntan, based on morphology traits and rDNA ITS min in a thermal cycler. The mix was then sequence. Riga et al. (2009) described yield centrifuged for 2 min at 14,000 r/min, the reduction of dryland peas and lentils caused by supernatant were used as template for PCR nematode, and the causal agent was identified as P. amplification. For replicates, 10 nematodes were hamatus by morphological traits. More recently, P. used to extract DNA, respectively. labiosus from different geographical areas in the For PCR amplification, a pair of universal continental United States were identified according primers, Pv1 (5’-ttgattacgtccctgcccttt-3’) and Pv2 to morphological and molecular characters (López (5’-tttcactcgccgttactaagg-3’) within the conserved et al., 2013). region (Vrain et al., 1992) of rDNA internal In the present study, we described pin transcribed space (ITS) sequence were used. PCR nematode P. lepidus from rizosphere of ramie. The were performed in a total volume of 15 ul nematode was identified with morphology traits containing 7.5 µl 2×masterMix (Beyotime, Nantong, combined with rDNA-ITS sequence. To our China), 0.6 µl each primer (10 mmol/l), 2.3 µl knowledge, this is the first report of P. lepidus sterilized double distilled water and 4 µl DNA. All parasitic on ramie. PCR reactions were performed in a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) with an Materials and methods initial denaturation at 95°C for 4 min, followed by Sampling and nematode extraction 34 cycles of 95°C for 45 sec, 50°C for 45 sec, 72°C Soils in 20 to 30 depths near the roots of for 1 min and a final extension of 10 min at 72°C. ramie planted in experimental fields in Yuangjiang, PCR products were analyzed with electrophoresis Hunan province were sampled. Nematodes from soil on 1.5% agarose gel. were extracted with the method as described by Anwar et al. (2012). Pin nematodes were picked Sequencing and analysis under a stereomicroscope and washed twice with Mini gel containing amplification fragments distilled water. The population was designated as were excised with a mobile UV light. The DNA YJ2012. fragments were purified from gel with a Universal DNA Purification Kit (Tiangen, Beijing, China) and Morphological study then cloned into pMD-18 vector (Takala, Dalian, Nematodes for morphological study were China) and later transformed into Escherichia coli heat killed, fixed in TAF and processed in slow DH5α competent cells (Tiangen, Beijing, China). SHORT COMMUNICATIONS 585 C SM V MB A B Fig. 1. Photomicrographs of female P. lepidus from Yuanjiang. A, whole body; B, Anterior region (MB, median bulb); C, Posterial region (SM, spermatheca; V, vulva). Bars: A-C, 50 µm. Transformed cells were placed onto LB plates Raski (1975) and of different population described containing ampicillin, X-gal and IPTG and by Chen et al. (2007), except for the a (body length / incubated at 37°C for overnight. After blue-white body width). Nematode YJ2012 is thinner than the selection, positive bacterial colonies identified by paratypes and Taiwan population. colony PCR with the primers as described above to Male: not found. exclude false positive. The resulted positive bacterial colonies were incubated in LB liquid Table I.- Comparison between the morphometrics of P. lepidus associated ramie and paratypes. containing ampicillin at 37°C for overnight. Recombinant plasmids were extracted with Plasmid Origin Characters +. purification mini kits (Tiangen, Beijing, China) and Yuanjiang Paratypes§. sequenced in Shanghai Sunny Biotechnology Co., Ltd. n 26 21 L 0.35±0.04 (0.26-0.42) -. 0.33 (0.28-0.40) a 18.46±2.22 (13.18-23.17) 25 (23-31) Results and discussion b 4.17±0.52 (3.39-5.14) 4.0 (3.4-4.6) Morphology c 12.87±2.1 (5.39-10.36) 25 (22-27) Female: After killed by gentle heat, female V 79.05±3.67 (18.09-84.94) 82 (80-84) body curved as “C” shape. The body is T 29.49±7.6 (19.53-38.89) 19.53-38.89 Sty 23.98±3.8 (13.73-27.91) 25 (22-27) characterized by obvious stylet, median bulb, vulva in posterior region and tapering tail (Fig. 1). Lip’s +.n, number; L, body length (mm); a, body length / maximum anterior end is flat and there is no annule. No clear width; b, body length / the distance from head end to posterior line between the posterior region and head was end of esophageal gland; c, body length / length of tail; V, ratio between distance from vulva to anterior end of body and total observed. Median bulb is oblong shape, with body length in %; T, tail length (µm); Sty, Stylet (µm). obvious valve gate. There are no clear division -.Measurements in form: mean ± standard deviation (range). between esophageal gland and intestine. Anus open §.Raski, D. J. (1975) is very small and not easy to see.