A c c e p t e d M a n u s c r i p t On-chip chemotaxis assay method of plant-parasitic nematodes was developed. A protocol to allow the nematodes to move around in the microchannels was achieved. Potassium nitrate stimulated the behavior of plant-parasitic nematode M. incognita. Potassium nitrate caused attraction or repulsion depending on its concentration. Abstract 25 Plant-parasitic nematodes substantially damage a wide variety of agricultural crops 26 worldwide. The chemotaxis of nematodes is a key factor in their parasitic relationship with 27 plants. Therefore, studies on nematode chemotaxis are essential for devising efficient and 28 environmentally friendly management methods. In this paper, we report a new, efficient, and 29 quantitative method to analyze chemotaxis of the plant-parasitic nematode, Meloidogyne 30 incognita, using a gel-filled microchannel device. We quantitatively defined time-dependent 31 concentration gradients of chemicals in the gel-filled microchannel by measuring changes in 32 the fluorescence intensity of fluorescein. We also developed a protocol to allow the 33 nematodes to move around in the microchannels by loading an agarose gel with optimum 34 concentration. Using this novel assay method, we have shown that potassium nitrate (KNO 3 ) 35 stimulates the behavior of M. incognita and can have both repellent and attractant effects, 36 depending on the concentration gradient. This newly developed quantitative chemotaxis assay 37 method can be used to screen and identify new candidate molecules that repel or attract 38 nematodes, and also to analyze the repellent/attractant properties of those molecules towards 39 nematodes. 40 41 42
Root-knot nematodes (Meloidogyne incognita) are one of the most economically damaging nematodes; thus it is important to know the molecular mechanisms involved in the phases of nematode infection. To use this nematode as a material for research, we must constantly prepare sufficient numbers of nematodes, preferably sterilized. Here we have developed alternative methods for culture, sterilization, and inoculation of root-knot nematodes that are especially suitable for the use of Arabidopsis thaliana Heynh as host plants. Nematol.
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This paper presents a novel PDMS (polydimethylsiloxane) device to analyze behaviors of plant-parasitic nematode, Meloidogyne incognita. For allowing the nematodes to swim in micro analyzed area, we developed a protocol of loading an agarose gel into microchannels by power-free pumping method. We experimentally confirmed that microchannel array 5 m wide, which is narrower than the body width of the nematodes, can efficiently confine the nematodes in analyzed area. Additionally, the microchannel array can generate chemical concentration gradients in the microchannel without external power sources when analyzing chemotaxis assay of the nematodes. We successfully demonstrated a chemotaxis assay of the nematode Meloidogyne incognita by using the developed on-chip device.
Meloidogyne incognita is one of the most detrimental root-knot nematode pests in the world because of its wide range of hosts, infecting almost all plant species. Following infection, this nematode induces gall formation in the root. We have found that these induced galls turn green in tomato roots exposed to light. This gall greening was light-dependent and inhibited by auxin treatment.Chlorophyll was detected in these green galls, and chloroplasts were also observed in the root-knot region. Expression of the chlorophyll alb-binding protein-4 gene was upregulated in the galls. These results indicate that light treatment induces chloroplast development in tomato plants infected with M. incognita during gall development.
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