水杨酸诱导鸭梨抗黑斑病效果及机理的探讨

doi:10.7668/hbnxb.20192811

摘要/Abstract

摘要：

为了揭示水杨酸诱导鸭梨抗黑斑病的作用效果及机理,以河北农业大学实验农场梨园鸭梨叶片和果实为试验材料,通过组织分离法分离、纯化梨黑斑病病原菌并对其进行致病性检测,利用形态观察及真菌 ITS、HIS、RPB2、ACT多基因综合鉴定法明确鸭梨黑斑病致病菌的类型;鸭梨离体叶片通过针刺接种黑斑病菌分生孢子悬浮液侵染0,6,12,24,48,72,96,120 h,通过实时荧光定量PCR检测水杨酸信号通路中相关基因的表达量变化,通过高效液相色谱(HPLC)检测0,72 h内源水杨酸含量的变化;利用不同浓度水杨酸(0,0.002,0.02,0.2,2.0,10.0,20.0 mmol/L)测定其对黑斑病菌菌丝生长的影响;采用外源0,0.02,0.2,2.0 mmol/L水杨酸处理鸭梨果实,接种黑斑病菌并观察其抗病效果。结果表明,河北农业大学实验农场梨园中鸭梨黑斑病病原菌为链格孢属链格孢菌,鸭梨离体叶片接种黑斑病菌后,72 h游离态水杨酸含量由0 mg/g增至0.02 mg/g,结合态水杨酸含量由0.47 mg/g增至1.55 mg/g,SA信号通路中相关基因Pbrgene12425、Pbrgene6286接种96 h与0 h相比表达量分别上调5.48,4.66倍,Pbrgene8895、Pbrgene43605接种120 h表达量上调7.90,10.0倍,外源施加0.2 mmol/L水杨酸显著提高鸭梨果实对黑斑病的抗性。在鸭梨抗黑斑病过程中,鸭梨离体叶片游离态水杨酸含量显著增加,水杨酸信号通路中相关基因被诱导上调表达,外源施加0.2 mmol/L水杨酸可提高鸭梨果实对黑斑病的抗性。

关键词: 鸭梨, 黑斑病, 链格孢菌, 水杨酸, 组织分离法, 荧光定量PCR

Abstract:

In order to reveal the effect and molecular mechanism of salicylic acid-induced resistance to black spot disease of Yali,the leaves and fruits of Yali were used as experimental material which were collected from the experimental orchard of Hebei Agricultural University.Tissue isolation method was used to isolate and purify the pathogen of black spot disease and detected its pathogenicity.Morphological observation and multi-genes of ITS,HIS,RPB2 and ACT were used to identify the pathogen of black spot disease.The detached leaves of Yali were inoculated with conidia suspension of Alternaria alternata by acupuncture for 0,6,12,24,48,72,96 and 120 h,respectively.Real-time RT-PCR was used to analyse the expression of related genes which involved in salicylic acid signaling,and high-performance liquid chromatography (HPLC) was used to detect the content of endogenous SA at 0 and 72 h,respectively.Exogenous SA treatment with different concentrations (0,0.002,0.02,0.2,2.0,10.0,20.0 mmol/L) were used to identify the effect to the mycelial growth of A.alternata;Exogenous SA treated Yali fruit with different concentrations (0,0.02,0.2,2.0 mmol/L),respectively,and inoculated with A.alternata to detect the effect of disease resistance.The results showed that the pathogen of Yali black spot disease was A.alternata. Detached leaves of Yali were inoculated with A.alternata for 72 h,the free state SA content increased from 0 mg/g to 0.02 mg/g and the binding state SA content increased from 0.47 mg/g to 1.55 mg/g,and for 96 h compared with 0 h,the expression of Pbrgene12425,Pbrgene6286, increased 5.48,4.66 times,respectively,the expression of Pbrgene8895 and Pbrgene43605 increased 7.90 and 10.0 times for 120 h.Exogenous 0.2 mmol/L SA significantly increased the resistance of Yali fruit to black spot disease.In the process of Yali resistance to black spot disease,the free state SA content of Yali leaves significantly increased,and the related genes in SA signaling were induced to up-expressed,exogenous 0.2 mmol/L SA significantly increased the resistance of Yali to black spot disease.

Key words: Yali, Black spot, Alternaria alternata, Salicylic acid, Tissue isolation method, qRT-PCR

魏亚蕊, 赵曙良, 成晓华, 闫琦, 刘娜, 张玉星. 水杨酸诱导鸭梨抗黑斑病效果及机理的探讨[J]. 华北农学报, 2022, 37(2): 183-191. doi: 10.7668/hbnxb.20192811.

WEI Yarui, ZHAO Shuliang, CHENG Xiaohua, YAN Qi, LIU Na, ZHANG Yuxing. Effect and Mechanism of the Disease Resistance to Pear Black Spot Induced by Salicylic Acid in Yali[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 183-191. doi: 10.7668/hbnxb.20192811.

http://www.hbnxb.net/CN/Y2022/V37/I2/183

图/表 8

表1 研究中所用引物序列

Tab.1 List of primer sequences used in this study

基因名称 Gener name	正向引物序列(5'—3') Forward primer sequence (5'—3')	反向引物序列(5'—3') Reverse primer sequence (5'—3')
ITS	TCCGTAGGTGAACCTGCGCGG	TCCTCCGCTTATTGATATGC
HIS3	ACTAAGCAGACCGCCCGCAGG	GCGGGCGAGCTGGATGTCCTT
ACT	TATCGTTCTCGACTCTGGTGAC	TCTGCATACGGTCGGAGATAC
RPB2	GGGGWGAYCAGAAGAAGGC	CCCATRGCTTGTYYRCCCAT
Pbrgene12425	AGGA TACACAGGGCTTTGGATTCG	AACACAGTGTACCCCCTCGG ATTC
Pbrgene6286	GCGGAGACATCTGAGTTTGC	CCAGGACTTCCGAGCAATGA
Pbrgene8895	CCCTGGAGCTAATAAACGTGG	CATGGAGATTAACGAGATGGATC
Pbrgene43605	GCAGCAGTAGGCGTTGGTCCCT	CCAGTGCTCATGGCAAGGTTTT

图1 鸭梨黑斑病田间症状和病原菌形态
A、B.鸭梨黑斑病病叶田间症状;C、D.病原菌菌落形态观察,比例尺为1 cm;E、F.病原菌分生孢子及分生孢子梗。

Fig.1 Field symptoms of Yali black spot disease and morphological characteristics of its pathogen
A,B.Conidia and conidiophore of pathogen;C,D.The field symptoms of Yali black spot;E,F.Morphological characteristics of fungal colonies,Bar is 1 cm.

图2 梨黑斑病病原菌HIS、ACT、RPB2、ITS基因的扩增及系统进化树
A.梨黑斑病病原菌HIS、ACT、RPB2、ITS基因的扩增:M.Marker。 B.梨黑斑病病原菌HIS、ACT、RPB2、ITS多基因系统进化树的构建:红色字体表示7种链格孢菌的HIS基因,蓝色字体表示的是6种链格孢菌的ACT基因,橙色字体代表8种链格孢菌的RPB2基因,粉红色字体代表8种链格孢菌的ITS基因;黑色字体梨黑斑Black spot HIS,梨黑斑Black spot ACT,梨黑斑Black spot RPB2,梨黑斑Black spot ITS分别代表鸭梨黑斑病病原菌的HIS,ACT,RPB2,ITS基因。

Fig.2 Amplification of HIS, ACT,RPB2 and ITS genes and phylogenetic tree of the pathogen of pear black spot disease
A.Amplification of HIS, ACT,RPB2 and ITS genes:M.Marker.B.Phylogenetic tree of pear black spot based on HIS,ACT,RPB2 and ITS multiple genes:The red font indicated the HIS gene in seven species of Alternaria,the blue font indicated the ACT gene in the six species of Alternaria,the orange font indicated the RPB2 gene in the eight species of Alternaria,the pink font indicated the ITS gene in the eight species of Alternaria,the black font black spot HIS,black spot ACT,black spot RPB2,black spot ITS indicated the HIS,ACT,RPB2,ITS gene in black spot of Yali.

图3 黑斑病菌侵染鸭梨叶片的症状
A.鸭梨离体叶片接种黑斑病菌后不同时间的病斑表型(比例尺为1.0 cm)。B.对鸭梨接黑斑病菌0,24~120 h共6个时间点病斑部位的放大。

Fig.3 Symptom development of Yali leaves inoculated with A.alternate infection
A.The disease spot phenotype of Yali leaves after inoculation at different times(Bar is 1 cm). B.Amplification of disease spots at 6 time points at 0,24-120 h after inoculation.

图4 黑斑病菌对鸭梨叶片内游离态水杨酸与结合态水杨酸含量的影响
样本间各小写字母表示不同时间点差异显著(P<0.05)。

Fig.4 The effect of the content of free and binding state SA in inoculated Yali leaves
Various letters among samples indicate significant difference at different time points(P<0.05).

图5 黑斑病菌侵染鸭梨叶片SA信号通路相关基因的表达

Fig.5 The expression of the related genes in SA signaling in inoculated Yali leaves

表2 外源不同浓度水杨酸对黑斑病菌菌丝生长的影响

Tab.2 The effect of different concentrations of SA on the growth of A.alternate

SA浓度/ (mmol/L) SA concentration	菌丝生长直径/cm Mycelium growth diameter	抑菌率/% Inhibitory rate
0	6.03±0.09a	0
0.002	5.48±0.14c	9.12
0.02	5.67±0.19ab	6.02
0.2	5.66±0.15bc	6.12
2.0	4.90±0.11d	18.78
10.0	4.26±0.26e	29.39
20.0	3.48±0.16f	42.27

图6 外施不同浓度水杨酸对鸭梨果实抗黑斑病的影响
A.不同浓度SA处理鸭梨接种黑斑病菌7 d的表型。B.不同浓度SA处理鸭梨接种黑斑病菌7,14 d病斑直径统计。比例尺为1 cm。样本间不同小写字母表示差异显著(P<0.05)。

Fig.6 The effect of exogenous different concentration of SA on resistance to black spot disease of Yali
A.The phenotype of pear fruits were treated with different concentration of SA after inoculation of A.alternate at 7 d.B.Data statistics of spot diameter of pear fruits treated with SA after inoculation with A.alternate at 7 and 14 d,respectively.Bar is 1 cm.Various letters indicate significant difference among samples (P<0.05).

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