Rust pathogen identification and mechanism of disease-resistance research on Kentucky bluegrass dwarf mutant
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摘要: 锈病是草坪草的重要病害，且病原菌种类多，鉴定困难。本研究以草地早熟禾野生型和矮化突变材料中的感病株系为对象，通过观察锈菌的形态特征，并结合ITS和β-tubulin基因序列分析，其中ITS序列的同源比对分析和β-tubulin基因的分子进化分析结果表明，该菌种与小麦禾柄锈菌的同源关系接近，所以初步认定该菌株属于禾柄锈菌，这是国内对禾柄锈菌引起草地早熟禾锈病的首次报道。同时，我们对病原菌诱导后草地早熟禾PR1L、NPR1L基因的表达变化和PRs蛋白表达进行了研究，发现PR1L、NPR1L基因在病菌诱导12 h时的表达量达到了峰值，且在矮化突变植株(A16)中的相对表达量分别达到了8.8-fold、4.5-fold，均大于在对照植株(WT)中的表达量。另外植物的PRs蛋白在禾柄锈菌侵染植物第8天后的表达量明显高于未侵染的植株。初步对草地早熟禾锈病的防御机制进行了探讨，为今后开展草地早熟禾锈病的预防及抗病育种研究奠定基础。Abstract: Rust is one of the destructive diseases on turfgrass. The species of rust pathogens are multiple and difficult to identify. In order to determine the rust pathogen species on Kentucky bluegrass wild type and dwarf mutant plant, pathogen morphology, along with ITS and β-tubulin gene sequence analysis were carried out. The analysis of ITS by BLAST and phylogenetic analysis of β-tubulin showed that the strains were related to Puccinia graminis of wheat, so we identified it as Puccinia graminis, which is the first report on Kentucky bluegrass in China. Moreover, transcriptional level of PR1L and NPR1L genes in Kentucky bluegrass inoculated with Puccinia graminis, as well as the expression of PRs protein, were investigated. The results presented that the expression of PR1L and NPR1L genes reached peak at 12 hours induced by Puccinia graminis, and the fold changes in dwarf mutant (A16) were 8.8 and 4.5, respectively, greater than in control wild type (WT). Besides, the expression of PRs in P. graminis infected plants 8 days later was higher than in non-infected plants. Defense mechanism of Kentucky bluegrass to rust was preliminary investigated and it presented the related disease-resistant gene and protein induced by rust pathogen. The research provides foundation for rust prevention and disease resistance breeding on Kentucky bluegrass in future.
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Keywords:
- turfgrass /
- Kentucky bluegrass /
- dwarf mutant /
- rust pathogen identification /
- disease resistance
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图 1 感病植株的发病症状和锈菌菌株夏孢子形态的光学显微镜观察
Figure 1. Symptoms of Kentucky bluegrass leaves infected by urediospore of rust and morphological investigation of the urediospore under optical microscope
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图 2 供试菌株及Puccinia相关菌株β-tubulin基因的系统发育树
候选锈菌菌株为供试材料的锈病病原菌。进化树右端的字母和数字代表GenBank中的序列号，分支上的数字代表自举的可信度，0.02为序列分歧度。
Figure 2. Phylogenetic tree of isolate strain and their relatives in Puccinia
Putative Puccinia is the isolate rust strain. Numbers in parentheses represent the sequences' accession number in Gen Bank. The number at each branch points is the percentage supported by bootstrap. Bar, 2% sequence divergence.
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图 3 草地早熟禾及其矮化突变材料受禾柄锈菌侵染后PR1L和NPR1L基因的表达变化情况
Figure 3. Expression pattern of PR1L, NPR1L induced by Puccinia graminis in Kentucky bluegrass wild type (WT) and its dwarf mutants (A16)
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图 4 草地早熟禾野生型(WT)及其矮化突变(A16)植株接种禾柄锈菌前后的病程相关蛋白电泳分析
1.蛋白marker；2.未接种的WT样品；3.未接种的A16样品；4.接种后的A16样品；5.接种后的WT样品。
Figure 4. 12% SDS-PAGE analysis of pathogenesis-related proteins extracted from leaves of WT and A16 inoculated with/without Puccinia graminis
1, protein marker; 2, the A16 sample inoculated without Puccinia graminis; 3, the WT sample inoculated without Puccinia graminis; 4, the A16 sample inoculated with Puccinia graminis; 5, the WT sample inoculated with Puccinia graminis.
表 1 实验所需引物的序列
Table 1 List of primer sequences used in this study
引物名称
Primer name引物序列
Primer sequence(5′—3′)ITS1 TCCGTAGGTGAACCTGCGG ITS4 TCCTCCGCTTATTGATATGC Tubulin-F ATGGAYTCGGTYCGATCTGGCG Tubulin-R CYTCTCCRGTGTACCAATGCAGG PR1L-qF CGCTACGCCCGCTCCC PR1L-qR GCCCCTCGTCCACCCA NPR1L-qF CAAGGAAGGGCAGACTAA NPR1L-qR GCAGCGATGTGAAGAACA Actin-qF TTGACTGAGAGGGGCT Actin-qR TCATACGGTCTGCGAT 
下载: 导出CSV
表 2 草地早熟禾的抗病相关的候选基因筛选
Table 2 List of candidate disease-resistant gene studied in Kentucky bluegrass clones
基因名称
Gene ID候选基因注释
Candidate gene annotation开放阅读框序列长度
ORF length/bp同源基因编号
GenBank accession of homologs同源物种
Allied speciesPR1L pathogenesis-related protein 1A-like 552 XM_004975168 栗Setaria italica NPR1L NPR1-like 1 protein 1 758 JX424315 硬粒小麦Triticum durum
下载: 导出CSV
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