Exogenous glycine betaine improved the resistance of apple to Glomerella leaf spot

Abstract:【Objective】Glomerella leaf spot (GLS), a fungal disease caused by G. cingulata, severely affects apple quality and yield. In the present experiment, we studied the effects of glycine betaine (GB) on the resistance of apple to G. cingulata, screened out the optimal exogenous concentration, and further analyzed its dynamic changes, so as to provide reference for the study of resistance mechanism of apple to G. cingulata.【Methods】GL-3 seedlings (Malus domestica‘Royal Gala-3’, provided by Prof. Zhihong Zhang at Shenyang Agricultural University, Shenyang, China) were used as plant materials. The optimal concentration of exogenous GB was screened by observing the phenotype, determining the lesion area, relative electrical conductivity (REC) and relative chlorophyll content (SPAD) in apple leaves after inoculation with G. cingulate (isolated and provided by Prof. Lili Huang at the College of Plant Protection, Northwest A & F University, Yangling, China). Furthermore, the oxidase activity, content of hormone and amino acid, and expression levels of pathogenesis- related genes in vitro apple leaves were analyzed to study the dynamic effects of exogenous GB (0, 1, 2, 4, 6 mmol· L^-1 ) on GLS. The determination of REC, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were all based on Junfeng Gao’s method. The relative chlorophyll content was measured by SPAD instru-ment. The diseased leaves were scanned by EPSON Chops V700 PHOTO scanner and the lesion area was calculated by ImageJ software. Hormones and amino acid were determined by liquid chromatography-mass spectrometry (LC-MS, LC: AC, ExionLC; MS: Qtrap5500 AB Sciex). The instrument used for real-time quantitative PCR was LightCycler® 96.【Results】After inoculation with G. cingulata, compared with 0 mmol·L^-1 concentration GB treatment, the lesion area of 1, 2, 4 mmol·L^-1 GB treatments decreased by 62.2%, 95.9% and 8.9%, respectively. While that of 6 mmol·L^-1 increased by 34.2%. The REC of the 1, 2, 4, and 6 mmol·L^-1 decreased by 21.1%, 88.4%, 35.2% and 2.0%, respectively. The SPAD value of 1, 2, 4 mmol·L^-1 increased by 0.8%, 28.7% and 5.4%, respectively. While the value of 6 mmol·L^-1 decreased by 7.0%. Summarely, 2 mmol·L^-1 GB treatment had the lowest lesion area, the lowest REC and the highest SPAD value. Then we further studied the effect of 2 mmol·L^-1 GB on the physiology of apple leaves after infection. The CAT activity treated with CK+GB, GLS, GLS+GB increased by 231.1%, 71.1% and 353.3%, the POD activity increased by 33.3%, 132.9% and 151.0%, and the SOD activity increased by 154.9%, 194.9% and 168.7% compared with the CK. The SA content treated with CK+GB, GLS and GLS+GB all basically increased with time. The SA content of CK+GB was always higher than that of the CK, and significantly increased by 1 513.7% on the 4th day of inoculation. The SA content of GLS+GB was always higher than that of the GLS, even significantly increased by 159.6% on the second day. JA content of CK + GB, GLS and GLS + GB increased first and then decreased at the early stage due to the antagonistic relationship between the JA and SA. The JA content of CK+GB reached the highest on the first day and significantly increased by 1 746.5% compared with the CK. The JA content of GLS+GB and GLS reached the peak on 0.5d, while that of the GLS+GB was significantly increased by 10.1% compared with the GLS. After 0.5 d, the contents of GLS+GB and GLS decreased, while that of the GLS increased again and then decreased after 2 d. These results indicated that GB could positively regulate the antagonistic relationship between the JA and SA after inoculation. The ABA and ACC contents of GLS and GLS+GB significantly increased compared with the CK, but the ABA and ACC contents of GLS+GB significantly decreased by 37.9% and 34.0%. The CK+GB had no significant difference. The content of IAA of GLS + GB, compared with GLS, first increased by 83.1% and then decreased by 55.0%. The CK+GB has no significant difference compared with the CK. The GLS and GLS + GB both induced a large amount of amino acid compared with the CK, and increased gradually over time. Except for 0 and 4 d, the amino acid content of GLS+GB was always significantly lower than that of the GLS, and decreased by 65.0%, 76.6%, 73.1% and 71.2% at 0.5, 1, 2 and 6 d, respectively. There was no significant difference in amino acid content between CK+GB and CK. The expression levels of the MdPR1, MdPR4, MdPR8, MdPAL were all up- regulated on 0.5-6 d, and the expression levels of chitinase and β-1, 3-glucanase genes were up-regulated at early stage, while the expression level of the MdERF3 had no significant difference.【Conclusion】Under the infection of G. cingulata, exogenous 2 mmol·L^-1 GB pretreatment could most effectively enhance the ability of plant cells to deal with the G. cingulata, increased the activity of the oxidase system, regulated the content of hormones, and enhanced the expression levels of pathogenesis-related genes, resulting in the improvement of the resistance of apple to G. cingulata .