Rice dwarf virus infection alters green rice leafhopper host preference and feeding behavior

Abstract

Host plants, pathogens and their herbivore vectors systems have complex relationships via direct and indirect interactions. Although there are substantial gaps in understanding these systems, the dynamics of the relationships may influence the processes of virus transmission and plant disease epidemics. Rice dwarf virus (RDV) is mainly vectored by green rice leafhoppers (GRLHs), Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae) in a persistently circulative manner. In this study, host plant selection preferences of non-viruliferous and viruliferous (carrying RDV) GRLHs between RDV-free and RDV-infected plants were tested. Non-viruliferous GRLHs preferred RDV-infected rice plants over RDV-free rice plants, and viruliferous GRLHs preferred RDV-free rice plants over RDV-infected rice plants. In odor selection preference bioassay using a four-field olfactometer, non-viruliferous GRLHs preferred odors of RDV-infected rice plants over healthy rice and viruliferous GRLHs preferred odors of RDV-free rice plants over RDV-infected ones. In 6 h plant penetration behavior bioassay using electrical penetration graphs, non-viruliferous GRLHs spent shorter time in non-penetration and much longer time in xylem feeding on RDV-infected, compared to RDV-free rice plants. Viruliferous GRLHs exhibited more salivation and stylet movement on RDV-free rice plants than on RDV-infected rice plants. We infer from these findings that RDV influences these vector behaviors by altering host plant physiology to promote viral transmission.

Fig 1. Non-viruliferous GRLHs preferred RDV-infected rice plants over RDV-free rice plants.

The histogram bars show numbers of insects settled on RDV-free and RDV-infected rice plants. The asterisks indicate statistical significance, determined by Chi-square test, with null hypothesis of no preference between the treatments (GRLHs not settled on any plant surface or died were not induced in the statistical analysis) [12] (* P < 0.05, * * P < 0.01).

Fig 2. Viruliferous GRLHs preferred RDV-free rice plants over RDV-infected rice plants.

The histogram bars show numbers of insects settled on RDV-free and RDV-infected rice plants. The asterisks indicate statistical significance, as determined by the Chi-square test, with null hypothesis of no preference between the treatments (GRLHs not settled on any plant surface or died were not induced in the statistical analysis) [12] (* P < 0.05, * * P < 0.01).

Fig 3. Odor selection preferences of non-viruliferous GRLHs exposed to rice plant volatiles.

Preferences were determined as times individual insects invested in each field of a four-field olfactometer during 1 h experiments. (A) RDV-free rice plants were placed in one odor source, with the other three fields charged with filtered clean air (n = 18). (B) RDV-infected rice plants were placed in one odor source, with the other three fields charged with filtered clean air (n = 15). (C) RDV-free and RDV-infected rice plants were placed in opposite fields (n = 20). The orientation of the fields is indicated relative to the field laced with the test odor: Non, RDV-free rice plants; RDV, RDV-infected rice plants; L, left; O, opposite; R, right. Scatter diagram show examples of location of single tracer GRLH per second during a 1 h experiment. The broken line at 900 s indicates an equal amount of time in all fields. Deviations from equal distribution were tested with a Friedman-ANOVA (P < 0.05). Bars annotated with different letters were significantly different from each other (Wilcoxon-Wilcox test).

Fig 4. Odor selection preferences of viruliferous GRLHs exposed to rice plant volatiles.

Preferences were determined as times individual insects invested in each field of a four-field olfactometer during 1 h experiments. (A) Same as Fig 3 panel A (n = 17). (B) Same as Fig 3 panel B (n = 18). (C) Same as Fig 3 panel C (n = 20). Scatter diagram show examples of location of single tracer GRLH per second during a 1 h experiment. The broken line at 900 s indicates an equal amount of time in all fields. Deviations from equal distribution were tested with a Friedman-ANOVA (P < 0.05). Bars annotated with different letters are significantly different from each other (Wilcoxon-Wilcox test).

Fig 5. Non-viruliferous GRLHs invested time in each waveform during 6 h recording periods.

The histogram bars show percentages of time invested in each waveform on RDV-free (n = 13) and RDV-infected (n = 15) rice plants. NP = non-penetration, Nc1 = penetration initiation, Nc2 = salivation and stylet movement, Nc4 = ingestion from phloem bundle tissues, Nc5 = ingestion from xylem bundle tissues. The data are means ± s. e. m. Histogram bars annotated with * indicate significant difference (P < 0.05), ns indicates no significant difference (P > 0.05).

Fig 6. Viruliferous GRLHs invested time in each waveform during 6 h recording periods.

The histogram bars show percentage of time invested in each waveform on RDV-free (n = 14) and RDV-infected (n = 17) rice plants. The data are means ± s. e. m. Histogram bars annotated with * indicate significant differences (P < 0.05), ns indicates no significant difference (P > 0.05).

Fig 7. Three EPG parameters of non-viruliferous GRLHs feeding on RDV- free (n = 13) and RDV-infected (n = 15) rice plants.

The data were electrically recorded during 6 h feeding periods on rice plants. The histogram bars show time (min) spent on each of the rice treatments. The data are means ± s. e. m. Histogram bars annotated with * were significantly different (P < 0.05).

Fig 8. Three EPG parameters of viruliferous GRLHs feeding on RDV- free (n = 14) and RDV-infected (n = 17) rice plants.

The data were electrically recorded during 6 h feeding periods on rice plants. The histogram bars show time (min) spent on each of the rice treatments. The data are means ± s. e. m. Histogram bars annotated with * were significantly different (P < 0.05).