广东省侵染美香占2号的稻瘟病菌致病性及无毒基因变异分析

doi:10.3864/j.issn.0578-1752.2022.07.007

Abstract

Abstract:

【Objective】The objective of this study is to analyze the pathogenicity and variation patterns of avirulence genes (Avr genes) genotype of Magnaporthe oryzae, which was collected from a high-quality rice variety Meixiangzhan 2 widely cultivated in Guangdong, and to provide a reference for the rational layout of Meixiangzhan 2 in different rice ecological areas. 【Method】The pathogenicity of single-spore strains was determined using 9 M. oryzae monogenic differentials. The DNA of the single-spore M. oryzae strains collected from Meixiangzhan 2 in different rice areas and in different years from 2013 to 2018 was subjected to PCR amplification using the functional markers of 8 Avr genes. The PCR products of Avr genes with promotor sequences or CDS regions were analyzed by agarose gel electrophoresis and the PCR products of representative strains were sequenced, and their sequences were compared to corresponding Avr genes, respectively. The corresponding M. oryzae monogenic differentials were used to determine the pathogenicity of M. oryzae strains with different mutation types of the Avr genes. According to two mating types MAT1-1 and MAT1-2 molecular markers of M. oryzae, the possible mating type of M. oryzae strains isolated from Meixiangzhan 2 was detected. 【Result】The pathogenicity analysis with a set of monogenic differentials showed that 52 rice blast strains collected from Meixiangzhan 2 from 2013 to 2018 were avirulent to IRBL9-W (Pi9), IRBLzt-T (Piz-t), NIL-e1 (Pi50) and IRBLkh-K3 (Pikh). The tested strains showed high frequencies (>57%) of virulence to IRBLz-Fu (Piz), IRBLkp-K60 (Pikp) and IRBLi-F5 (Pii), and the frequency of virulence to IRBLz-Fu (Piz) showed significant differences in different years, suggesting that the M. oryzae population in the field of Meixiangzhan 2 planted area had a higher frequency of virulent strains of avrPiz, avrPikp and avrPii. AvrPiz-t, AvrPi9 and AvrPik-CDE fragments were almost present in all tested strains, but none of Avr1-CO39, AvrPia or AvrPii was amplified in any strain. Only 4 strains could amplify the fragment of AvrPita, and only 3 strains could amplify the fragment of AvrPik-ABF. Amplicon sequencing of the 7 strains revealed that the sequences of AvrPi9 and AvrPiz-t were identical to those of the corresponding Avr genes. Of the 7 sequenced strains in AvrPik-CDE, 4 strains were almost identical to each other, and 3 strains had a ‘CTTT' in the coding region of AvrPik. Four different amplification types of AvrPib (expected size bands, bands with transposon Pot3 insertion, bands with transposon Mg-SINE insertion, and non-amplified bands) were detected by electrophoresis analysis and 5 different haplotypes (AvrPib-AP1-1, AvrPib-AP1-2, avrPib-AP2, avrPib-AP3 and avrPib-AP2+avrPib-AP3) were detected by sequencing PCR products. The genotypes AvrPib-AP1-1 and AvrPib-AP1-2 were avirulent, while avrPib-AP2 and avrPib-AP3 showing insertions of transposon Pot3 or Mg-SINE with different insertion sites were virulent. A double haplotype of avrPib-AP2+avrPib-AP3 was detected in one strain. The result of mating type analysis with molecular markers showed that all of 52 tested strains belonged to the mating type of MAT1-2, two strains (GD18-009 and GD18-185) from Shaoguan, Guangdong were also detected with MAT1-1 mating type, indicating that these two strains had dual mating types. 【Conclusion】Among the strains from the high-quality rice variety of Meixiangzhan 2 in Guangdong, the Avr genes of AvrPiz-t, AvrPi9, AvrPi50 and AvrPikh distribute widely. The variation types of AvrPib are abundant. The results of this study will provide useful information for the rational layout of Meixiangzhan 2 and the deployment of other rotation varieties with different genotypes of rice blast resistance.

Key words: Meixiangzhan 2, Magnaporthe oryzae, genotype of avirulence gene, mating type

WANG WenJuan,SU Jing,CHEN Shen,YANG JianYuan,CHEN KaiLing,FENG AiQing,WANG CongYing,FENG JinQi,CHEN Bing,ZHU XiaoYuan. Pathogenicity and Avirulence Genes Variation of Magnaporthe oryzae from a Rice Variety Meixiangzhan 2 in Guangdong Province[J].Scientia Agricultura Sinica, 2022, 55(7): 1346-1358.

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References 40

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采集地 Sampling location	菌株编号 Strain number	采集地 Sampling location	菌株编号 Strain number
韶关Shaoguan	GD13-012	韶关Shaoguan	GD16-187
韶关Shaoguan	GD13-027	韶关Shaoguan	GD16-189
韶关Shaoguan	GD13-354	韶关Shaoguan	GD16-196
韶关Shaoguan	GD13-358	韶关Shaoguan	GD17-001
韶关Shaoguan	GD13-369	河源Heyuan	GD17-125
韶关Shaoguan	GD13-382	惠州Huizhou	GD17-165
韶关Shaoguan	GD13-389	惠州Huizhou	GD17-171
韶关Shaoguan	GD13-394	惠州Huizhou	GD17-181
韶关Shaoguan	GD13-395	韶关Shaoguan	GD17-312
河源Heyuan	GD13-452	韶关Shaoguan	GD17-313
阳江Yangjiang	GD13-591	韶关Shaoguan	GD17-314
河源Heyuan	GD14-073	惠州Huizhou	GD17-380
韶关Shaoguan	GD14-186	韶关Shaoguan	GD18-009
云浮Yunfu	GD14-295	惠州Huizhou	GD18-094
清远Qingyuan	GD15-074	韶关Shaoguan	GD18-185
清远Qingyuan	GD15-076	韶关Shaoguan	GD18-188
惠州Huizhou	GD15-097	韶关Shaoguan	GD18-189
韶关Shaoguan	GD15-195	韶关Shaoguan	GD18-191
韶关Shaoguan	GD15-196	韶关Shaoguan	GD18-192
韶关Shaoguan	GD15-198	韶关Shaoguan	GD18-193
韶关Shaoguan	GD15-205	韶关Shaoguan	GD18-194
韶关Shaoguan	GD15-206	韶关Shaoguan	GD18-196
韶关Shaoguan	GD15-214	惠州Huizhou	GD18-269
韶关Shaoguan	GD16-006	惠州Huizhou	GD18-271
河源Heyuan	GD16-034	惠州Huizhou	GD18-272
韶关Shaoguan	GD16-185	增城Zengcheng	GD18-361

引物 Primer	引物序列 Sequence (5′-3′)	预期片段大小 Expected fragment size (bp)	目的 Purpose
Avr1-CO39 F	TGCCGCATTTTGCTAACCG	994	检测Avr1-CO39启动子及CDS To detect Avr1-CO39 promoter and CDS
Avr1-CO39 R	GCGAATCCATAGACAAGGAC	994	检测Avr1-CO39启动子及CDS To detect Avr1-CO39 promoter and CDS
AvrPita F	CAGGCATACATTGGAGAGCC	1549	检测AvrPita启动子及CDS To detect AvrPita promoter and CDS
AvrPita R	CCCTCCATTCCAACACTAAC	1549	检测AvrPita启动子及CDS To detect AvrPita promoter and CDS
AvrPii F	GGTAGATATCCGCTGACTGG	839	检测AvrPii启动子及CDS To detect AvrPii promoter and CDS
AvrPii R	ACTGTCCGCCGCTCGTTTGG	839	检测AvrPii启动子及CDS To detect AvrPii promoter and CDS
AvrPi9 F	ATGCAGTTCTCTCAGATCCTC	342	检测AvrPi9 CDS To detect AvrPi9 CDS
AvrPi9 R	CTACCAGTGCGTCTTTTCGAC	342	检测AvrPi9 CDS To detect AvrPi9 CDS
MGG-AvrPiz-t F	AACCCGCAGCAGCATAAAAG	1621	检测AvrPiz-t启动子及CDS To detect AvrPiz-t promoter and CDS
MGG-AvrPiz-t R	GAAAAGTGGCTCGTTCCTAATTG	1621	检测AvrPiz-t启动子及CDS To detect AvrPiz-t promoter and CDS
AvrPia F	CAGAGAAACGGACTTGGAGG	1220	检测AvrPia启动子及CDS To detect AvrPia promoter and CDS
AvrPia R	GGTATACACGTACGGTAGGG	1220	检测AvrPia启动子及CDS To detect AvrPia promoter and CDS
AvrPik-CDE F	TCCTGCTGCTAACTCCATTC	~1000	检测AvrPik-CDE型启动子及CDS To detect AvrPik-CDE promoter and CDS
AvrPik-CDE R	GGGTACAGGAATACCAGG	~1000
AvrPik-ABF F	TCCTGCTGCTAACTCCATTC	~1000	检测AvrPik-ABF型启动子及CDS To detect AvrPik-ABF promoter and CDS
AvrPik-ABF R	GGGTACAGGAATATCAGC	~1000
MGG-AvrPib F2	GGCCTATCGCATGTTTATATTGAG	633	检测AvrPib启动子及部分CDS To detect AvrPib promoter and some CDS
MGG-AvrPib R2	AGGCGATAGTGATAAAAGTGGTTG	633
MAT1-1F	GACATCAAGGCTCGTCGAC	650	交配型分析 Mating type analysis
MAT1-1R	GAGTGCGCCATTAGGAAGC	650	交配型分析 Mating type analysis
MAT1-2F	GGACCTCGAATTCGTCGAC	300	交配型分析 Mating type analysis
MAT1-2R	GGCATCTTTCAGGATAGACC	300	交配型分析 Mating type analysis

年份（菌株数） Year (Number of strains)	IRBL9-W	IRBLzt-T	NIL-e1	IRBLkh-K3	IRBL1-CL	IRBLta2-Re	IRBLz-Fu	IRBLkp-K60	IRBLi-F5	CK
年份（菌株数） Year (Number of strains)	Pi9	Piz-t	Pi50	Pikh	Pi1	Pita²	Piz	Pikp	Pii	LTH
2013 (11)	0	0	0	0	0	9.09	81.82	81.82	72.73	100.00
2015 (9)	0	0	0	0	0	33.33	88.89	88.89	77.78	100.00
2016 (6)	0	0	0	0	16.67	16.67	100.00	66.67	83.33	100.00
2017 (9)	0	0	0	0	33.33	22.22	77.78	88.89	77.78	100.00
2018 (14)	0	0	0	0	14.29	21.43	57.14	78.57	78.57	100.00
χ² for homogeneity					59.61***	15.25**	12.37**	4.16	0.73

			AvrPib等位基因的核苷酸多态性Nucleotide polymorphisms of AvrPib alleles^a
			-269 bp	-266 bp/-232 bp/-228 bp/-52 bp	-216 bp	-124 bp	-71 bp
单倍型 Haplotype	频率 Frequency (%)	表型 Pathotype to IRBLb-B (Pib)	- - - -		-	- - -
AvrPib-AP1-1	20.00	A	- - - -		C	AAC
AvrPib-AP1-2	6.67	A	TAAC		C	AAC
avrPib-AP2	33.33	V	- - - -或TAAC	Pot3
avrPib-AP3	33.33	V	- - - -		C	AAC	Mg-SINE
avrPib-AP2+avrPib-AP3	6.67	V	- - - -	Pot3	C	AAC	Mg-SINE

Pathogenicity and Avirulence Genes Variation of Magnaporthe oryzae from a Rice Variety Meixiangzhan 2 in Guangdong Province

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