Induction of resistance to diseases in plant by aerial ultrasound irradiation

Abstract

Ultrasound, which refers to frequencies above the audible limit of human hearing, is a candidate for inducing resistance to pathogens in plants. We revealed that aerial ultrasound of 40.5 kHz could induce disease resistance in tomatoes and rice when the plants were irradiated with ultrasound of ca. 100 dB for 2 weeks during nursery season and reduced the incidence of Fusarium wilt and blast diseases, respectively, when plants were inoculated with pathogen 0 or 1 week after terminating irradiation. Disease control efficacy was also observed with ultrasound at frequencies of 19.8 and 28.9 kHz. However, cabbage yellows and powdery mildew on lettuce were not suppressed by ultrasound irradiation. No significant positive or negative effect on growth was observed in tomato and rice plants. RT-qPCR showed that the expression of PR1a involved in the salicylic acid (SA) signaling pathway was upregulated in the ultrasound-irradiated tomato.

Keywords: SA signaling pathway; blast; physical control; powdery mildew; soilborne fusarium disease; ultrasound.

Fig. 1. Aerial ultrasonic conditions and an experimental flow. Seedlings of each plant were irradiated with ultrasound of 40.5 kHz frequency with ca. 100 dB sound pressure level. The oscillation pattern was composed of intermittent pulse waves. The pulse width is 7 msec, and the pulse frequency repeatedly shifts. The intermittent pulse waves are illustrated (a). Irradiation with ultrasound started when plants were 1 week old, continued for 1–2 weeks (24 hr a day), and plants were inoculated with pathogens at 0 or 1 week after terminating ultrasound irradiation (b). Tomato seedlings irradiated with aerial ultrasound in a limited irradiation area (50 cm diameter at 70 cm from the oscillator) in a sound-proof area (c).

Fig. 2. Wilt was suppressed in tomato plants irradiated by aerial ultrasound before inoculation. One-week-old tomato seedlings were irradiated with 40.5 kHz ultrasound for 1 or 2 weeks (I) or without irradiation (NI) and inoculated with the tomato wilt pathogen F. oxysporum f. sp. lycopersici (Fol) 0 or 1 week after terminating ultrasound irradiation. About 40 days after inoculation, the severity of each plant’s disease was evaluated from 0 to 4 following the indexes: 0, no symptoms; 1, lower leaves yellowing; 2, lower and upper leaves yellowing; 3, lower leaves yellowing and wilting and upper leaves yellowing; 4, all leaves wilting and yellowing or dead. Two-week ultrasound-irradiated tomato plants (cv. Moneymaker) presented significant wilt suppression when they were inoculated with Fol just after the termination of ultrasound irradiation (0 wpui) (a). Two-week ultrasound-irradiated tomato plants (cv. Moneymaker) presented wilt suppression when they were inoculated with Fol 1 week after the termination of ultrasound irradiation (1 wpui) (b). One week (1w) of irradiation also conferred a wilt control effect in cv. Moneymaker when plants were inoculated with Fol 1 week after the termination of ultrasound irradiation, but the effect of 2-week (2w) irradiation was higher (c). The severity of disease was analyzed with R, Wilcoxon rank sum test. p<0.05, bars=standard error, Asterisk means significance as compared to NI.

Fig. 3. Wilt was suppressed in tomato plants irradiated by aerial ultrasound using the traveling device where the total period of irradiation of each plant by ultrasound was as low as 1/6 of the time as compared to that with the fixed device. Tomato plants were irradiated with 40.5 kHz ultrasound for 2 weeks for 15 sec at 75-sec intervals for 2 weeks (I) or without irradiation (NI). Tomato wilt was suppressed the same as with continuous 2-week irradiation. The tomato wilt disease index was analyzed with R, Wilcoxon rank sum test. p<0.05, bars=standard error, Asterisk means significance as compared to NI.

Fig. 4. Disease suppression by ultrasound irradiation possibly differs depending on the plant species or plant-pathogen interactions. One-week-old tomato seedlings (cv. Momotaro) were irradiated with 40.5 kHz ultrasound for two weeks and inoculated with tomato powdery mildew pathogens just after terminating ultrasound irradiation. Two weeks after inoculation, the disease incidence of each plant was graded from 0 to 4 using the following index: 0, no symptoms; 1, a foliar area of 0–5% indicating powdery mildew symptoms; 2, 6–30%; 3, 31–60%; 4, 61–100% (a). One-week-old rice seedlings (cvs. Aichi-asahi and Kinuhikari) were irradiated with ultrasound for two weeks and inoculated with rice blast pathogen (Po) just after terminating ultrasound irradiation. One week after inoculation, the lesions on the upper three leaves of each plant were counted (b). One-week-old cabbage seedlings were irradiated with ultrasound for two weeks and inoculated with cabbage yellows pathogens just after terminating ultrasound irradiation. Two weeks after inoculation, the disease incidence of each plant was graded from 0 to 4 using the following index: 0, no symptoms; 1, lower leaves yellowing; 2, lower and upper leaves yellowing; 3, lower leaves yellowing and wilting and upper leaves yellowing; 4, all leaves wilting and yellowing or dead (c). The disease indexes of tomato wilt and cabbage yellows were analyzed with R, Wilcoxon rank sum test, and the number of lesions of rice blasts was analyzed with R, t-test. p<0.05, bars=standard error, Asterisk means significance as compared to NI.

Fig. 5. The heights of tomato and rice plants did not change with ultrasound wave irradiation. We examined the effects of ultrasound on plant growth. The heights of tomato (cv. Momotaro) and rice (cv. Aichi-asahi) plants did not show differences as compared to NI plants. The heights of tomato and rice plants were analyzed with R, t-test. p<0.05, bars=standard error.

Fig. 6. The expression of PR1a possibly involved in the SA signaling pathway was upregulated in tomato plants irradiated by ultrasound. Analyses of the expression of defense-related genes were performed by RT-qPCR. Genes suggested to be involved in the SA signaling pathway—PAL1, NIM1, and PR1a—and a gene involved in the JA-signaling pathway—LoxD—were examined. PR1a was upregulated in leaves at 0 hpi, and LoxD was downregulated in leaves and stems at 0 hpi. Total RNA was isolated from leaves (white circle), stems (light gray), and roots (dark gray) of tomato plants (cv. Moneymaker) irradiated with ultrasound (40.5 kHz, ca. 100 dB, intermittent pulse wave, 2 weeks) at 0 and 24 hr post inoculation (hpi). The relative expression levels of mRNA were determined by normalizing the PCR threshold cycle number of each gene with that of the Actin2 reference gene. Three biological replicates were used in the experiments. The relative expression levels of the mRNA of I were compared to those of NI and analyzed with R, t-test. *, p<0.10; **, p<0.05, bars=standard error.