Differences in resistance to Pseudomonas syringae pv. actinidiae and acting mechanism of different kiwifruit varieties

Abstract: 【Objective】The bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae is a severe threat to kiwifruit production. The disease, with a truly high pandemic proportion, has caused huge economic loss in many countries. A lot of researchers found that resistance breeding was a safe and effective method to control kiwifruit bacterial canker. However, only a few studies have been focused on the mechanism of kiwifruit resistance to bacterial canker so far. According to the differences in physiological changes between resistant and susceptible varieties after inoculation, we can reveal the mechanism of kiwifruit against Pseudomonas syringae pv. actinidia, and provide a theoretical basis for controlling kiwifruit bacterial canker.【Methods】We evaluated nine kiwifruit varieties using quantitative inoculation in vitro. The resistant variety(Actinidia deliciosa‘Xuxiang') and susceptible variety(A. chinensis‘Hongyang') were used to investigate the relative expression levels of resistance genes PR1, PR5, POD and PAL, and investigate the dynamic changes of the activity of defense enzymes including catalase(CAT), peroxidase(POD), superoxide dismutase(SOD) and phenylalanine ammonia lyase(PAL).【Results】Of the 9 kiwifruit varieties that were tested in this study, 25 days later, the results showed that there was a significant difference in the size of lesions among the varieties after they were inoculated with Psa, and no any species was immune to kiwifruit bacterial canker. According to the size of the lesions, the degree of disease showed,‘Hongyang'>‘Xixua'>'Huayou',‘Cuixiang',‘Jinyan'>‘Yate',‘Qinmei'>‘Hayward'>‘Xuxiang', the lesion of‘Hongyang'was 15.99 cm, and the lesion of‘Xixuan'was 1.76 cm.‘Xuxiang'was proved to be a resistance genotype with a low incidence of infection, however,‘Hongyang'was highly susceptible. After‘Xuxiang'and‘Hongyang'varieties were inoculated with Psa, we compared the process of disease spot spreading. The results showed that, the symptoms of‘Hongyang'appeared at the earliest time, and water-stained lesions were visible under the epidermis 10 days after inoculation, while Xu Xiang's lesions appeared at the latest time, and after 15 days of inoculation, slight lesions could be seen. After 25 days, the lesions of two varieties were stable, no longer expanded, and became dry. Then, we measured the expression level of genes that related to defense enzymes, and the Real-time quantitative PCR results displayed, the relative expression level of these genes were up-regulated in resistant variety‘Xuxiang', and higher than the susceptible variety‘Hongyang'. The expression level of PR1 gene increased gradually during the whole infection in both varieties. It was up-regulated by 92.52 times on 12 th day with‘Xuxiang'and50.76 times on 14 th day with‘Hongyang'. The expression trend of PR5 gene in two varieties was the same as that of PR1 gene, and the difference was obvious. The relative expression level in‘Xuxiang'was particularly pronounced, with the highest increase on the 12 th day, up-regulated by 61.54 times,and the highest on the 14 th day in‘Hongyang', up-regulated 14.78 times. As the genes that could regulate the activity of the corresponding enzymes, the PAL and POD were up-regulated at 2 th day and 4 th day, respectively in‘Xuxiang', but in‘Hongyang'they were up-regulated at 10 th day after inoculation, and the relative expression level was low. The relative expression levels of CAT and SOD genes were not significantly different between the two varieties after they were inoculated with Psa, and there was no obvious correlation with the corresponding enzyme activities. We also found that the activities of four defense enzymes were significantly enhanced in both resistant and susceptible varieties compared with uninfected groups. Except for SOD, the CAT, POD and PAL showed a trend of“first increase and then decrease”. The change of enzyme activities of‘Xuxiang'was particularly prominent,and the enzyme activities reached peaks before symptoms occurred, which significantly inhibited the expansion of lesions. However, the activities of the enzymes at the early stage of infection were low in‘Hongyang', and the expansion rate of the lesion was very fast, and the enzyme activities reached the maximum at the late stages of the disease. Among them, the peak activity of PAL and POD enzymes was significantly different,‘Xuxiang'was significantly higher than‘Hongyang', and the peak appeared earlier than‘Hongyang'. The activities of PAL enzyme increased rapidly in‘Xuxiang', reaching the peak at 5 th day after inoculation, and the enzyme activity was 2.31 times compared with uninfected group. However, the enzyme activity reached a peak at 20 th day with‘Hongyang', and the enzyme activity was 1.8 times more than the uninfected group. The POD activity of‘Xuxiang'first reached the peak at 5 th day after inoculation, which was 1.66 times more than the uninfected Psa group,and at 20 th day it reached the peak again and was 1.81 times higher than the uninfected. But POD showed a slow growth trend in‘Hongyang'variety, reached peak at 20 d after inoculation, and the enzyme activities were very lower than that in‘Xuxiang'. Although the SOD activity was significantly enhanced, there was no significant difference between the two varieties. For CAT, the enzyme activity of‘Xuxiang'only showed a small peak in the early stage of the disease and was higher than that of‘Hongyang', and subsequently, the changes in CAT activity were small. The activity of CAT in‘Hongyang'showed an increasing trend and maintained higher activities during the whole infection period, the enzyme activity was much higher than that of‘Xuxiang', which reached the peak at 20 d after inoculation, which was 7.34 times more than that of the uninfected group.【Conclusion】After evaluation of disease-resistant varieties, it was indicated that‘Xuxiang'can be used as an ideal material for disease-resistant breeding, PR1 and PR5 genes can be used as effective markers for kiwifruit resistant variety.PAL and POD activities can be used as one of the biochemical markers for screening resistant plants.