Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (10): 1899-1907.doi: 10.3864/j.issn.0578-1752.2018.10.009

• PLANT PROTECTION • Previous Articles     Next Articles

Influence of Temperature and Polyamines on Occurrence of Citrus Canker Disease and Underlying Mechanisms

Feng YANG1(), ChuanWu CHEN2(), QiJun FAN2, ChunMei SHI1, ZongZhou XIE1, DaYong GUO1, JiHong LIU1()   

  1. 1College of Horticulture and Forestry Sciences, Huazhong Agricultural University/Key Laboratory of Horticultural Plant Biology, Ministry of Education, Wuhan 430070
    2Guangxi Academy of Specialty Crops/Guangxi Key Laboratory of Citrus Biology, Guilin 541004, Guangxi
  • Received:2017-10-16 Accepted:2017-12-06 Online:2018-05-16 Published:2018-05-16

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Abstract:

【Objective】 Canker disease is one of the most devastating diseases that cause serious damages to citrus. It is more likely to occur under high temperature. The objective of this study is to elucidate the mechanism underlying the disease incidence at high temperatures, reveal its metabolic changes, and to provide important theoretical guidance for controlling the disease using certain chemicals.【Method】 Sweet orange (Citrus sinensis), which is sensitive to canker disease, was used as the experimental material. The sweet orange plants were pre-cultured for 3 d at either 21℃ or 30℃ prior to inoculation with Xanthomonas citri subsp. citri (Xcc), followed by evaluation of disease incidence. Expression of four defense-related genes, including AOS (allene oxide synthase), CHI (chitinase), GPX (glutathione peroxidase) and PR4A (pathogenesis-related protein 4A), in the plants pre-cultured at the two temperatures was assessed by semi-quantitative RT-PCR. Meanwhile, endogenous polyamines (putrescine, spermidine and spermine) in the plants pre-cultured at the two temperatures were also analyzed by HPLC. In addition, sweet orange plants were treated with exogenous spermidine (0.4 mmol·L-1), using water treatment as a control, before Xcc inoculation. Disease incidence and index of plants treated with either spermidine or water were compared, while endogenous polyamine contents and expression levels of defense-related genes (AOS, CHI, GPX and PR4A) in response to spermidine or water treatment were assessed. 【Result】 After inoculation with Xcc, it was found that plants pre-cultured at 21℃ exhibited a lower cankder disease incidence at the early stage when compared with the plants pre-cultured at 30℃. On the 10th day, the incidence of the two treatments was similar. HPLC analysis showed that content of the three free polyamines (putrescine, spermidine and spermine) in plants pre-cultured at 21℃ was significantly higher than that in the plants pre-cultured at 30℃. In addition, RT-PCR analysis indicated that the transcript level of three defense-related genes, CHI, GPX and PR4A, in plants kept at 21℃ was higher than that from 30℃, while there was no significant difference in AOS expression between the two groups. Exogenous application of spermidine remarkably enhanced levels of endogenous putresicne and spermidine, reduced disease incidence and index in comparison with water treatment. Spermidine treatment reduced the disease incidence by 45% and in comparison with the control after 14 days of inoculation. In addition, the disease index of the spermidine-treated samples was 4.8 lower than that of the control. Meanwhile, the phenotype indicated that the control displayed more serious symptom than that of spermidine treatment. Moreover, spermidine treatment could up-regualte mRNA abundances of all four defense-realted genes, including AOS, CHI, GPX and PR4A. 【Conclusion】 Sweet orange displayed susceptibility to citrus canker at high temperature, and the potential mechanisms underlying this phenomenon may be ascribed to inhibition of defense-related genes and suppression of polyamine biosynthesis. Exogenous polyamine treatment conferred enhanced tolerance to citrus canker by upregulating defense-related genes and triggering disease resistance response. Taken together, high temperature is one of the environmental factors accounting for outbreak of citrus canker disease, and polyamines are conducive for improving tolerance to citrus canker disease.

Key words: citrus, canker disease, high temperature, polyamines, defense-related genes, disease resistance

Table 1

HPLC elution procedures for separation of free polyamines"

时间
Time (min)		 A相
A phase (%)		 B相
B phase (%)
0 45 55
15 95 5
17 100 0
18 45 55
25 45 55

Table 2

Primer sequences used for RT-PCR analysis in this study"

基因
Gene		 引物序列Primer sequence (5′-3′)
正向引物Forward primer 反向引物Reverse primer
CHI TCTTGCCCTAGCTTTTCCCAC GCAATCTCACGCTTC GAAACTT
AOS ATTCCACCTACACGGAGGCAT TAAC GGAGCGAGCTGAAACAG
GPX CAGTGTGGCTTGACCAATTCAA CCCCTCCTTATCCACCAAGAAC
PR4A ACATAACTGTAGTGCCCATGAGC GGAGGCTTAGATTTGGACGAAGG
Actin ATCCCTCAGCACCTTCC CCAACCTTAGCACTTCTCC

Fig. 1

Canker disease incidence of Xcc-inoculated plants that have been pre-cultured at 21℃ or 30℃ for three days"

Fig. 2

Free polyamine contents (A) and expression levels of defense-related genes (B) in plants pre-cultured at 21℃ or 30℃ for three days"

Fig. 3

Free polyamine levels (A) and expression levels of defense-related genes (B) in plants treated with exogenous spermidine or water"

Fig. 4

Effects of exogenous spermidine treatment on canker disease resistance"

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