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A novel in vitro chemotaxis bioassay to assess the response of Meloidogyne incognita towards various test compounds

Tagginahalli N. Shivakumara
Tagginahalli N. Shivakumara
, 
Tushar K. Dutta
Tushar K. Dutta
 and 
Uma Rao
Uma Rao
   | Dec 03, 2018
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Published Online: Dec 03, 2018
Page range: 487 - 494
DOI: https://doi.org/10.21307/jofnem-2018-047
Keywords
© 2018 Tagginahalli N et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Attraction of M. incognita J2s toward tomato root tip in PF-127 medium in a petri dish. (A) 50 J2s were inoculated at 1.5 cm distance (marked by black dot) from the tomato root tip. (B) Numerous sinusoidal tracks inscribed on PF-127 medium due to J2 locomotion towards tomato root were documented.
Attraction of M. incognita J2s toward tomato root tip in PF-127 medium in a petri dish. (A) 50 J2s were inoculated at 1.5 cm distance (marked by black dot) from the tomato root tip. (B) Numerous sinusoidal tracks inscribed on PF-127 medium due to J2 locomotion towards tomato root were documented.

Figure 2

Actual photograph of an in vitro chemotaxis assay plate used in the present study. Due to the premixing of pink colored acid fuchsin stain with PF-127 gel, areas containing agar and PF-127 can be easily demarcated. Nematode inoculation point (*) is 1.5 cm equidistant from agar-PF-127 junction. Agar-PF-127 junction is 1 cm distant from the nearest edge of the petri plate. In well A, different test compounds were applied to observe either nematode attraction or repulsion. In well B, diluents (in which test compounds were dissolved) were applied as control.
Actual photograph of an in vitro chemotaxis assay plate used in the present study. Due to the premixing of pink colored acid fuchsin stain with PF-127 gel, areas containing agar and PF-127 can be easily demarcated. Nematode inoculation point (*) is 1.5 cm equidistant from agar-PF-127 junction. Agar-PF-127 junction is 1 cm distant from the nearest edge of the petri plate. In well A, different test compounds were applied to observe either nematode attraction or repulsion. In well B, diluents (in which test compounds were dissolved) were applied as control.

Figure 3

A standard curve displays absorbance (550 nm) calibration values plotted against different concentrations of acid fuchsin (1, 5, 10, 15, 20, and 25 µl) in PF-127 gel which were fit to a linear correlation model (R 2 = 0.98).
A standard curve displays absorbance (550 nm) calibration values plotted against different concentrations of acid fuchsin (1, 5, 10, 15, 20, and 25 µl) in PF-127 gel which were fit to a linear correlation model (R 2 = 0.98).

Figure 4

Qualitative (A) and quantitative (B) analyses of establishment of concentration gradient of a test chemical (acid fuchsin) in chemotaxis assay plate. Intensity of color suggests that acid fuchsin had diffused from its point source to nematode inoculation point by 40 min. Concentration of acid fuchsin (absorbance at 550 nm) attained an equilibrium at 40 min and remained in steady-state up to 4 hr at different indicated distances from the point source. Error bars indicate the standard error between three biological and technical replicates.
Qualitative (A) and quantitative (B) analyses of establishment of concentration gradient of a test chemical (acid fuchsin) in chemotaxis assay plate. Intensity of color suggests that acid fuchsin had diffused from its point source to nematode inoculation point by 40 min. Concentration of acid fuchsin (absorbance at 550 nm) attained an equilibrium at 40 min and remained in steady-state up to 4 hr at different indicated distances from the point source. Error bars indicate the standard error between three biological and technical replicates.

Figure 5

The chemotactic response of M. incognita J2 to volatile test compounds. (A) Chemotaxis bioassays show attraction (positive index) or repulsion (negative index) values. Statistical analysis performed using one-way ANOVA with Tukey’s HSD (p values: *p < 0.05; **p < 0.01; ***p < 0.001) tests comparing with water as negative control. Five µl of test compounds were screened at six different dilutions (100, 10−1, 10−2, 10−3, 10−4, and 10−5) against approximately 100 J2s. Error bars represent standard error of three biological and three technical replicates. (B) A close-up view of the assay plate shows that greater number of J2s was accumulated at agar-PF-127 junction in the odorant side compared to lesser number in the diluent side while exposed to isoamyl alcohol and 2-butanone.
The chemotactic response of M. incognita J2 to volatile test compounds. (A) Chemotaxis bioassays show attraction (positive index) or repulsion (negative index) values. Statistical analysis performed using one-way ANOVA with Tukey’s HSD (p values: *p < 0.05; **p < 0.01; ***p < 0.001) tests comparing with water as negative control. Five µl of test compounds were screened at six different dilutions (100, 10−1, 10−2, 10−3, 10−4, and 10−5) against approximately 100 J2s. Error bars represent standard error of three biological and three technical replicates. (B) A close-up view of the assay plate shows that greater number of J2s was accumulated at agar-PF-127 junction in the odorant side compared to lesser number in the diluent side while exposed to isoamyl alcohol and 2-butanone.

Figure 6

The chemotactic response of M. incognita J2 to host root exudates. Chemotaxis bioassays show attraction (positive index) or repulsion (negative index) values. Statistical analysis performed using one-way ANOVA with Tukey’s HSD (p values: *p < 0.05; **p < 0.01; ***p < 0.001) tests comparing with water as negative control. Ten microliters of neat exudates was tested against approximately 100 J2s. Error bars represent standard error of three biological and three technical replicates.
The chemotactic response of M. incognita J2 to host root exudates. Chemotaxis bioassays show attraction (positive index) or repulsion (negative index) values. Statistical analysis performed using one-way ANOVA with Tukey’s HSD (p values: *p < 0.05; **p < 0.01; ***p < 0.001) tests comparing with water as negative control. Ten microliters of neat exudates was tested against approximately 100 J2s. Error bars represent standard error of three biological and three technical replicates.
eISSN:
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Language:
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Journal Subjects:
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