CC BY
28Russian Journal of Nematology, 2012, 20 (2), 171-172
Short Note
Comparison of methods for culturing the reniform nematode, Rotylenchulus reniformis
Bie Yun Tsai and Huei Zhen Huang
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan,
e-mail: bieyntm@ntu.edu.tw
Accepted for publication 12 June 2012
Summary. The culture of Rotylenchulus reniformis in the liquid Gamborg's B5 medium yielded the highest amount, of 103,389 nematodes per dish, where the solid Gamborg's B5 medium produced 68,371 nematodes per dish. Both methods were more successful than culturing on plants grown in sand. The culture pouches method had the lowest nematode yield. The liquid medium has the advantage to the solid medium in easier handling of egg-mass picking. Key words: Culture, excised root, medium, Rotylenchulus reniformis.
Accepted for publication 22 August 2012
The reniform nematode, Rotylenchulus reniformis Linford & Oliveira, is an important nematode pest of many crops in many countries (Siddiqi, 1972; Robinson, 2007). The conventional way of culturing the reniform nematode is to grow it on host plants in soil in pots. This method is laborious and needs plenty of space. Space saving methods, such as using culture pouches (Tsai, 2008a), callus tissue ( Lownsbery et al., 1967) or excised roots (Tsai, 2001), have been developed for culturing Meloidogyne incognita and Pratylenchus species. However, information in regard to the suitability of these methods for culturing R. reniformis is lacking.
The purpose of our study was to compare the following methods for culturing the reniform nematode: (1) culturing on plants grown in culture pouches; (2) culturing on excised roots in Gamborg's B5 medium (GIBCOBRL) with 0.8% agar; and (3) culturing on excised roots in liquid medium, Gamborg's B5 medium without agar. Culturing the reniform nematode on plants grown in sand was used as control.
The reniform nematode was originally isolated from the experimental station of the National Taiwan University and cultured on mung bean Vigna radiata (L.) Wilczek. Individuals of a young female and male were picked under a dissecting microscope in the lamellar flow and surface sterilised in H2O2 for 2 minutes (Tsai, 2008b) and then inoculated onto the excised tomato roots grown
in Gamborg's B5 medium with 0.8% agar. The sterile population was sub-cultured every 7 weeks and maintained in our laboratory as the source of sterile inoculums. The nematodes were extracted with autoclaved modified Baermann funnels with lids under sterile condition. Approximately 10,000 nematodes of mixed stages with 18.8% young females were used as inoculums for each test. Tomato roots were cut from water agar plates and transferred to Gamborg's B5 medium with or without 0.8% agar in Petri dishes. Mung bean seeds were surface sterilised and planted in culture pouches (Tsai, 2008a). Another batch of surface-sterilised mung bean seeds were planted in sterile sand in pots. They were kept at 28°C in a growth chamber. The nematodes were inoculated 7 days later for all the treatments. The nematodes were extracted with a modified Baerman funnel 6 weeks after inoculation. There were four replicates for each treatment. The experiment was repeated twice and showed similar results. The data from two experiments were pooled.
The resulted nematode yields are shown in Fig. 1. The liquid Gamborg's B5 medium yielded the highest amount of R. reniformis, 103,389 nematodes per dish, although not significantly different from the yield of solid Gamborg's B5 medium, 68,371 nematodes per dish. Both Gamborg's B5 culture methods yielded more nematodes than in control. The culture pouches method had the lowest nematode yield.
Bie Yun Tsai and Huei Zhen Huang
Fig. 1. The effect of different culture methods on the yield of Rotylenchulus reniformis. Bars with different letters are significantly different at P<0.05 according to Duncan's multiple range test.
Apart from the higher nematode yield, the culture of the reniform nematode in Petri dishes, whether in the liquid or solid Gamborg's B5 medium, provides the opportunity to observe the development of the nematodes and production of egg masses The culture of Rotylenchulus reniformis in the liquid Gamborg's B5 medium yielded the highest amount, of 103,389 nematodes per dish, where the solid Gamborg's B5 medium produced 68,371 nematodes per dish. Both methods were more successful than culturing on plants grown in sand. The culture pouches method had the lowest nematode yield. The liquid medium has the advantage to the solid medium in easier handling of egg-mass picking. which is difficult in the sand culture. The liquid medium has the advantage to the solid medium in easier egg-mass picking. It is important where egg masses are used as experimental material. Ko & Van Gundy (1988) have used Pluronic F127 polyol, which liquefies at low temperature, as an alternative to agar. However, Pluronic F127 polyol has toxic effect on some nematodes. Eyre & Caswell (1991), when culturing R. reniformis, liquefied gellan gum by adding disodium ethylenediamin-etetraacetate (EDTA). However, EDTA has been reported to be an environmental pollutant (Yuan & Van Briesen, 2006). The method of culturing the reniform nematode in Gamborg's B5 medium used in this study is simpler and lacks the abovementioned side effects.
REFERENCES
Eyre, M. J. & Caswell, E. P. 1991. Sterile culture of Rotylenchulus reniformis on tomato root with gellan gum as a supporting medium. Journal of Nematology 23: 229-231.
Ko, M. P. & Van Gundy, S. D. 1988. An alternative gelling agent for culture and studies of nematodes, bacteria, fungi, and plant tissues. Journal of Nematology 20: 478-485.
Lownsbery, B. F., Huang, C. S., & Johnson, R. N. 1967. Tissue culture and maintenance of the root-lesion nematode, Pratylenchus vulnus. Nematologica 13: 390-394.
Robinson, A. F. 2007. Reniform in U.S. cotton: when, where, why, and some remedies. Annual Review of Phytopathology 45: 263-288.
Siddiqi, M. R. 1972. Rotylenchulus reniformis. C.I.H. Descriptions of plant-parasitic nematodes. Set 1, No. 5. Commonwealth Institute of Helminthology, England.
Tsai, B.Y. 2001. Effect of nematode age and plant species on the penetration speed of Meloidogyne javanica and Pratylenchus coffeae. Plant Pathology Bulletin 10: 65-70.
Tsai, B. Y. 2008a. Recycled papers as supporting substrates for root-knot nematode culture. Plant Pathology Bulletin 17: 65-68.
Tsai, B. Y. 2008b. Competition between Pratylenchus coffeae and Meloidogyne incognita. Plant Pathology Bulletin 17: 271-278.
Yuan, Z., & Van Briesen, J. M. 2006. The formation of intermediates in EDTA and NTA biodegradation. Environmental Engineering Science 23: 533-544.
