Effect and Mechanism of the Disease Resistance to Pear Black Spot Induced by Salicylic Acid in Yali

doi:10.7668/hbnxb.20192811

Abstract

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

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.

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Figures/Tables 8

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

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.

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.

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.

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).

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

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

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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