Sclerospora graminicola inhibited the growth and disrupted panicle development of foxtail millet
Downy mildew occurs in the whole growth period of foxtail millet and the symptoms from the germination to heading stage are different and special. Seeds of foxtail millet infected by Sclerospora graminicola may fail to germinate or the seedlings died before emergence. The abaxial surface of infected leaves displayed grey mildew which is called “grey back” (Fig. 1A). In addition, Sclerospora graminicola caused abnormal development of panicles of foxtail millet. Some disease plants did not produce spike at heading time and an etiolated leaf grew at the top plants instead. This kind of leaves was called “white tip” (Fig. 1B). With the senility of host plants, the“white tip” leaves turned brown and split longitudinally which looked like hair (“white hair”, Fig. 1C). Some disease plants produced hedgehog-like panicles which was the result of inflorescences was replaced by leaves (Fig. 1D).
Low concentration of exogenous SA enhanced the disease resistance of foxtail millet
SA can improve the resistance to pathogens of some plants, we hypothesized that application of exogenous SA may enhance the resistance of foxtail millet to downy mildew. To prove this, we planted seeds of xiaomi (an Arabidopsis-like mini foxtail millet) mixed with oospores of Sclerospora graminicola. Three-leaf stage seedlings were sprayed with different concentrations of SA were used to spray the leaves to explore the effect of different SA concentrations on the downy mildew of foxtail millet, and the incidence of disease was investigated after heading stage. The ratio of plants displayed disease phenotype including grey back, white tip, white hair and hedgehog-like panicles were counted. The result showed that with the increase of SA concentration, the incidence of foxtail millet after spraying treatment showed a decreasing-increasing-decreasing trend (Fig. 2). The incidence of foxtail millet treated with 1 mM to 3 mM SA spray was lower than the control (0 mM SA). The incidence of disease under 1 mM SA spray treatment was significantly lower than other treatments, and the incidence rate decreased by 24.26% compared with 0 mM SA spray treatment. On the contrary, high concentration SA treatment did not reduce disease incidence, but made foxtail millet more susceptible to downy mildew.
Exogenous SA inhibits the growth and development of foxtail millet
Exogenous SA reduced the disease incidence, we tested whether SA affect the growth and development of Jingu 21 (JG21) and GBS, two different varieties of foxtail millet. JG21, a cultivar with the largest planting area in China, is very susceptible to downy mildew. GBS was another variety with high resistance to downy mildew. Through incidence survey in our experimental field, we found the average incidence in last 3 years of JG21 was 10 times higher than GBS (Supplemental Table 2).
In this study, the resistant variety GBS and susceptible variety JG21 were cultured in Hoagland Solution, and different concentrations of SA were sprayed at the three-leaf stage. It was found that SA treatment had inhibition on the growth of foxtail millet. With the increase of SA concentration, the overall trend of plant height, fresh weight and dry weight of both JG21 and GBS were downward (Supplemental Table 3). To compare the differences between the two varieties, we set the control group at 100% and analyzed the rate of growth change after SA treatments. The results showed that SA with low concentration (1 mM and 3 mM) reduced the growth plant height, fresh weight and dry weight of of foxtail millet, and the change rate of susceptible variety JG21 was higher than resistant variety GBS. SA with high concentration (6 mM and 9 mM) seriously suppressed the growth of foxtail millet, however, there was not significant different of the change rate between the two varieties (Fig. 3).
Effects of SA on chlorophyll content of foxtail millet
Chlorophyll is an important material in plant photosynthesis, whose content is closely related to the photosynthetic rate of plants. The content of chlorophyll is positively related to the net photosynthetic rate and it can also be used as one of the important index to reflect the growth status of foxtail millet seedlings. Data above indicated that the SA concentration was 1 mM to 3 mM to induce the disease resistance of foxtail millet and we wondered the effect of different concentrations of SA spraying on the chlorophyll content. We sampled at 6 time points before treating (0 h) and spraying after 3, 6, 9, 12, and 24 h, the chlorophyll content of leaves was measured and the relative content of chlorophyll was calculated (the ratio of chlorophyll content at 3, 6, 9, 12, 24 h and 0 h). The results indicated that with the application of 1 mM to 3 mM SA spraying, the chlorophyll content in foxtail millet leaves had the same trend, which showed a decrease-increase-decrease. At each time point after treatment, chlorophyll content in leaves at 3 mM SA was significantly higher than that at 1 mM SA (Fig. 4). The results showed that the appropriate concentration of SA could promote the accumulation of chlorophyll in seedlings, thereby affecting the growth of foxtail millet.
Exogenous SA affects the content of malondialdehyde, proline, soluble sugar and the activity of phenylpropanoid in foxtail millet
In order to reveal the reason why SA improves disease resistance, we examined some substances related to plant resistance. Malondialdehyde (MDA) is the final product of cell membrane lipid peroxidation, which is toxic to cells, damages cell biofilms and can inhibit the activity of protective enzymes. Therefore, the degree of plant damage can be judged by MDA content. As shown in Fig. 5A, the relative content of MDA in untreated plants showed an upward trend over time. The MDA content in foxtail millet treated with 1 mM or 3 mM SA was increased compared with 0 mM when 3–9 h after treatment. But at 24 h after treatment, the MDA content of 1 mM-3 mM SA spray treatment was significantly lower than that of 0 mM SA treatment.
Soluble sugar mainly participates in the respiration metabolism of organisms by providing energy for life activities. Soluble sugar can improve the osmotic regulation ability of plant cells and reduce the damage degree of plasma membrane under stresses. We found that the soluble sugar content of foxtail millet in 0 mM SA and 3 mM SA showed decreasing-increasing-decreasing trend with the processing time increases, while the change of soluble sugar content under 1mM SA was opposite. Under 3 mM SA treatment, the soluble sugar content increased significantly compared with 0mM treatment at 3 ~ 6 h and 12 ~ 24 h, and the maximum increased about 3 times (12 h). At 9 h after treatment, the soluble sugar content in foxtail millet treated with 3 mM SA was the lowest, and significantly lower than 0 mM treatment. The soluble sugar content of 1mM SA treatment increased significantly at 6 h ~ 12 h compared with 0 mM treatment, and the maximum increased about 1.6 times (Fig. 5B).
Proline (PRO) contribute to the stability of normal metabolism of plants under stress by regulating osmotic balance, scavenging reactive oxygen species and reducing the toxic substances. At the early stage of SA spraying treatment (3 h-6 h), the relative content of PRO was significantly increased when the SA concentration was 3 mM compared with the 0 mM treatment, while the relative content of PRO at the later stage of the treatment (9 h-24 h) was significantly lower than that in the 0 mM treatment; PRO relative content in the 1 mM SA treatment was significantly lower than that in the 0 mM treatment at different time points after spraying; with the prolongation of the treatment time, the change trend of the PRO content under the three SA concentration treatments was basically the same (Fig. 5C).
Phenylpropanoid metabolism is an important pathway for the metabolism of secondary metabolites in plants, and the changes in enzyme activity of this pathway are closely related to plant disease resistance. In this study, we found that the phenylalanine ammonia lyase (PAL) activity in foxtail millet showed an overall decreasing-increasing-declining trend under 1 mM and 3 mM SA treatment, while the change of PAL activity under 0 mM SA treatment was opposite. With the increase of treatment time, PAL activity under 3 mM SA treatment was significantly lower than that under 0 mM SA treatment (Fig. 5D).
Exogenous SA modulated the expression level of resistance related genes in foxtail millet
To reveal the reason why SA improves disease resistance, we also tested the expression level of some resistance related genes. POD1, POD2 and SOD encode peroxidase (POD) and superoxide dismutase (SOD) which are the key enzymes in the oxygen free radical scavenging system. The expression levels of SiPOD1 and SiPOD2 in foxtail millet seedlings treated with 1 mM to 3 mM SA tended to be consistent with the prolongation of treatment time, showing an increase-decrease-increase. The expression levels of the two genes showed a turning point at 3 h and 9 h after treatment. At 3–24 h after treatment, the expression of SiPOD1 treated with 1 mM-3 mM SA was significantly up-regulated; and the expression of SiPOD2 was also significantly up-regulated after treatment with 1 mM SA, and the expression of this gene was the highest at 3 h after treatment, which was higher than that before treatment (0 h) increased by about 35.6 times (Fig. 6A-6B). The expression of SiSOD was significantly up-regulated at 3–12 h after treatment with 1 mM-3 mM SA compared with the before treatment (0 h), especially at 9–12 h, the expression of SiSOD treated with 3 mM SA was significantly increased compared with the other two concentrations; with the prolongation of treatment time, the expression trend of SiSOD gene was consistent, which showed that it first increased and then decreased (Fig. 6C).
PR1 gene is a downstream gene involved in the process of plant acquired resistance, and is involved in the process of MAPK regulating plant homeostasis. Studies have shown that SA can affect the expression of PR1. In this study, SiPR1 gene expression was detected by different concentrations SA leaf spraying. The results showed that the expression of SiPR1 gene in 1mM SA treated foxtail millet seedlings showed an increase-decrease-increase-decrease trend. However, after 3mM SA treatment, the change of gene expression was opposite, showing a decreasing-increasing-decreasing trend. The gene expression level after 1 mM and 3 mM SA treatment was significantly higher than that before treatment (0 h), but decreased after 24 h treatment (Fig. 6D).
SiPAL1 and SiPAL2 are key genes of PAL pathway which is necessary for plant resistance. Treating seedlings with 1mM SA could significantly increase the expression levels of SiPAL1 and SiPAL2 at the early stage (3 h-6 h). While it was opposite with 3 mM SA. the expression levels of SiPAL1 and SiPAL2 were significantly decreased at 3 h after treatment (Fig. 6E-6F).