Control Effect of Ralstonia Phage RPZH6 Strain on Tobacco Bacterial Wilt and Its Complete Genome Analysis

doi:10.13304/j.nykjdb.2021.0891

Abstract

Abstract:

Crops bacterial wilt is a bacterial soil-borne and vascular bundle disease in the world which is severely harmful and difficult to control. Choosing the Ralstonia solanacearum TBRS12 as host bacterium， bacteriophage RPZH6 was isolated from tobacco rhizosphere soil using double-layer plate method. The pot experiment was used to identify its biological control effect， and whole genome sequencing， comparative genome and phylogenetic tree were used to analyze the genomic structural characteristics and evolutionary relationship. The results showed that the control effect of phage RPZH6 on tobacco bacterial wilt could reach 60.98% at 21 d after inoculation， and the control effect was also 53.85% at 35 d after inoculation， which was significantly higher than that with chemical treatment of CuOH. Nucleic acid identification and whole genome analysis showed that the genome of phage RPZH6 was double-stranded DNA with a total length of 64 657 bp and GC content of 64.84%. It contained 92 open reading frames with 1 tRNA， and the coded products of 47 genes were predicted to be functional and others were putative unknown protein. Comparative genome and phylogenetic analysis showed that phage RPZH6 was a novel member of the Bcep22-like subfamily of the family Podoviridae. In conclusion， this paper cleared the biological control effect， genomic structural characteristics and evolutionary relationship of the bacteriophage RPZH6， and the results provided theoretical basis for the further application and analyzing control mechanism.

Key words: bacteriophage, Ralstonia solanacearum, genome analysis, biological control

摘要：

作物青枯病是世界范围的土传维管束细菌性病害，为害重、防治难。以烟草青枯菌TBRS12为宿主菌，采用双层平板法从烟草根际土壤中分离获得一株裂解性噬菌体RPZH6。采用室内盆栽法测定噬菌体RPZH6的生防效果，并通过全基因组测序、比较基因组及系统发育树进行鉴定分析。结果表明，接种后21 d，噬菌体RPZH6对烟草青枯病的防治效果为60.98%；接种后35 d，防治效果为53.85%，显著高于化学药剂氢氧化铜处理。核酸鉴定及全基因组分析显示，噬菌体RPZH6的基因组为双链DNA，全长64 657 bp，GC含量为64.84%，含有92个开放阅读框，1个tRNA，其中47个基因的编码产物为注释功能蛋白，其余为功能未知的假定蛋白。比较基因组及系统发育树分析显示，噬菌体RPZH6是一株新的短尾噬菌体科、Bcep22家族成员。上述结果为该噬菌体的进一步开发应用及生防机制探讨提供了理论基础。

关键词: 噬菌体, 青枯雷尔氏菌, 基因组分析, 生物防治

CLC Number:

S476

Zhijian LIN, Changjiang CHEN, Ting ZHOU, Gang GU, Fangping HU, Chunying LI, Xueqing CAI. Control Effect of Ralstonia Phage RPZH6 Strain on Tobacco Bacterial Wilt and Its Complete Genome Analysis[J]. Journal of Agricultural Science and Technology, 2022, 24(10): 133-142.

林志坚, 陈长江, 周挺, 顾钢, 胡方平, 李春英, 蔡学清. 青枯菌噬菌体RPZH6株系对烟草青枯病的生防效果及全基因组测序分析[J]. 中国农业科技导报, 2022, 24(10): 133-142.

Figures/Tables 6

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处理 Treatment	7 d		14 d		21 d		28 d		35 d
处理 Treatment	病情指数Disease index	防治效果Control effect/%	病情指数Disease index	防治效果Control effect/%	病情指数Disease index	防治效果Control effect/%	病情指数Disease index	防治效果Control effect/%	病情指数Disease index	防治效果Control effect/%
RPZH6+TBRS12	4.17 b	57.10 a	19.44 c	58.83 a	22.22 b	60.98 a	30.56 b	55.10 a	33.33 c	53.85 a
Cu（OH）₂+TBRS12	5.55 b	42.90 a	36.11 b	23.53 b	54.17 a	4.86 b	62.50 a	8.17 b	65.28 b	9.61 b
TBRS12	9.72 a	—	47.22 a	—	56.94 a	—	68.06 a	—	72.22 a	—
CK	0.00	—	0.00	—	0.00	—	0.00	—	0.00	—

处理 Treatment	7 d		14 d		21 d		28 d		35 d
处理 Treatment	病情指数Disease index	防治效果Control effect/%	病情指数Disease index	防治效果Control effect/%	病情指数Disease index	防治效果Control effect/%	病情指数Disease index	防治效果Control effect/%	病情指数Disease index	防治效果Control effect/%
RPZH6+TBRS12	4.17 b	57.10 a	19.44 c	58.83 a	22.22 b	60.98 a	30.56 b	55.10 a	33.33 c	53.85 a
Cu（OH）₂+TBRS12	5.55 b	42.90 a	36.11 b	23.53 b	54.17 a	4.86 b	62.50 a	8.17 b	65.28 b	9.61 b
TBRS12	9.72 a	—	47.22 a	—	56.94 a	—	68.06 a	—	72.22 a	—
CK	0.00	—	0.00	—	0.00	—	0.00	—	0.00	—

开放阅读框ORF	开放阅读框位置 Position of ORF/bp	蛋白大小 Protein size/aa	链 Strand	起始密码子 Start codon	功能预测 Possible function
1	1～262	86	+	GTG	转录调节蛋白 Putative transcription regulator protein
2	296～478	60	-	ATG	含甘氨酸拉链2TM结构域的蛋白质 Glycine zipper 2TM domain-containing protein
4	1 069～1 542	157	-	ATG	病毒粒子相关蛋白 Virion associated protein
5	1 620～2 729	369	-	ATG	主要衣壳蛋白 Major capsid protein
6	2 831～3 283	150	-	ATG	含AP2结构域蛋白AP2 domain-containing protein
7	3 328～3 525	65	-	ATG	碳储存调节器 Carbon storage regulator
11	4 844～7 189	781	-	GTG	门脉蛋白 Portal protein
14	8 170～9 915	581	-	ATG	末端酶大亚基蛋白 Terminase large subunit protein
16	10 578～10 769	63	-	ATG	辅酶A酯裂解酶 CoA ester lyase
17	10 769～10 867	32	-	ATG	类连接蛋白 Connectin-like protein
24	13 090～13 326	78	-	ATG	转录调节因子 Transcriptional regulator
32	15 480～15 725	81	-	GTG	末端酶小亚基蛋白 Terminase small subunit protein
34	16 836～17 324	162	-	ATG	HNH归巢核酸内切酶Ⅱ HNH homing endonuclease Ⅱ
41	19 861～20 667	268	-	ATG	DnaC类蛋白 DnaC-like protein
42	20 667～21 437	102	-	TTG	复制蛋白 Replication protein
46	24 667～25 026	119	-	ATG	DUF1364家族蛋白 DUF1364 family protein
50	26 150～26 731	193	-	ATG	单链DNA结合蛋白 Single-stranded DNA-binding protein
52	27 410～27 778	122	-	ATG	转录调节因子 Transcriptional regulator
53	27 805～28 497	230	+	ATG	转录调节因子 Transcriptional regulator
54	28 570～29 016	148	+	TTG	转录调节因子 Transcriptional regulator
55	29 183～30 211	342	+	ATG	RecB类核酸内切酶 RecB-like endonuclease
56	30 250～31 203	317	+	ATG	RecT类蛋白酶 RecT-like protein
57	31 209～31 436	75	+	ATG	DNA结合蛋白 DNA-binding protein
58	31 461～31 907	148	+	ATG	DNA结合蛋白 DNA-binding protein
59	31 932～32 996	354	+	ATG	DNA聚合酶Ⅲ β亚基 DNA polymerase Ⅲ subunit beta
61	34 378～34 650	90	+	ATG	Pyocin激活蛋白 PrtN Pyocin activator protein PrtN
63	35 571～36 764	397	+	ATG	酪氨酸重组酶 Tyrosine recombinase
64	36 838～37 005	55	+	TTG	酪氨酸重组酶 Tyrosine recombinase
65	37 017～37 553	178	-	ATG	Rz类裂解蛋白 Rz-like lysis protein
67	37 929～38 309	126	-	ATG	趋化蛋白 Chemotaxis protein
69	38 626～39 291	221	-	TTG	含DUF3380结构域蛋白 DUF3380 domain-containing protein
72	40 455～46 457	2 000	-	ATG	DarB类抗抑制蛋白 DarB-like antirestriction protein
73	46 436～50 134	1 232	-	GTG	DarB类抗抑制蛋白 DarB-like antirestriction protein
74	50 192～51 736	514	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
75	51 733～52 458	241	-	ATG	PAPS还原酶类蛋白 PAPS reductase-like protein
76	52 470～53 303	277	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
77	53 293～53 850	185	-	ATG	酰基辅酶A N-酰基转移酶 Acyl-CoA N-acyltransferase
79	54 246～55 979	577	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
80	55 991～56 977	328	-	ATG	尾纤蛋白 Tail fiber protein
81	57 063～58 382	439	-	ATG	推定尾纤蛋白 Putative tail fiber protein
82	58 382～58 663	93	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
84	60 366～61 343	325	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
85	61 350～61 772	140	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
88	62 505～63 167	220	-	ATG	病毒粒子相关蛋白 Virion associated protein
90	63 713～64 150	145	-	GTG	DarB类抗抑制蛋白 DarB-like antirestriction protein
91	64 147～64 512	121	-	GTG	钼蝶呤结合蛋白 Molybdopterin-binding protein
92	64 534～64 657	40	-	TAT	细胞色素B Cytochrome B

开放阅读框ORF	开放阅读框位置 Position of ORF/bp	蛋白大小 Protein size/aa	链 Strand	起始密码子 Start codon	功能预测 Possible function
1	1～262	86	+	GTG	转录调节蛋白 Putative transcription regulator protein
2	296～478	60	-	ATG	含甘氨酸拉链2TM结构域的蛋白质 Glycine zipper 2TM domain-containing protein
4	1 069～1 542	157	-	ATG	病毒粒子相关蛋白 Virion associated protein
5	1 620～2 729	369	-	ATG	主要衣壳蛋白 Major capsid protein
6	2 831～3 283	150	-	ATG	含AP2结构域蛋白AP2 domain-containing protein
7	3 328～3 525	65	-	ATG	碳储存调节器 Carbon storage regulator
11	4 844～7 189	781	-	GTG	门脉蛋白 Portal protein
14	8 170～9 915	581	-	ATG	末端酶大亚基蛋白 Terminase large subunit protein
16	10 578～10 769	63	-	ATG	辅酶A酯裂解酶 CoA ester lyase
17	10 769～10 867	32	-	ATG	类连接蛋白 Connectin-like protein
24	13 090～13 326	78	-	ATG	转录调节因子 Transcriptional regulator
32	15 480～15 725	81	-	GTG	末端酶小亚基蛋白 Terminase small subunit protein
34	16 836～17 324	162	-	ATG	HNH归巢核酸内切酶Ⅱ HNH homing endonuclease Ⅱ
41	19 861～20 667	268	-	ATG	DnaC类蛋白 DnaC-like protein
42	20 667～21 437	102	-	TTG	复制蛋白 Replication protein
46	24 667～25 026	119	-	ATG	DUF1364家族蛋白 DUF1364 family protein
50	26 150～26 731	193	-	ATG	单链DNA结合蛋白 Single-stranded DNA-binding protein
52	27 410～27 778	122	-	ATG	转录调节因子 Transcriptional regulator
53	27 805～28 497	230	+	ATG	转录调节因子 Transcriptional regulator
54	28 570～29 016	148	+	TTG	转录调节因子 Transcriptional regulator
55	29 183～30 211	342	+	ATG	RecB类核酸内切酶 RecB-like endonuclease
56	30 250～31 203	317	+	ATG	RecT类蛋白酶 RecT-like protein
57	31 209～31 436	75	+	ATG	DNA结合蛋白 DNA-binding protein
58	31 461～31 907	148	+	ATG	DNA结合蛋白 DNA-binding protein
59	31 932～32 996	354	+	ATG	DNA聚合酶Ⅲ β亚基 DNA polymerase Ⅲ subunit beta
61	34 378～34 650	90	+	ATG	Pyocin激活蛋白 PrtN Pyocin activator protein PrtN
63	35 571～36 764	397	+	ATG	酪氨酸重组酶 Tyrosine recombinase
64	36 838～37 005	55	+	TTG	酪氨酸重组酶 Tyrosine recombinase
65	37 017～37 553	178	-	ATG	Rz类裂解蛋白 Rz-like lysis protein
67	37 929～38 309	126	-	ATG	趋化蛋白 Chemotaxis protein
69	38 626～39 291	221	-	TTG	含DUF3380结构域蛋白 DUF3380 domain-containing protein
72	40 455～46 457	2 000	-	ATG	DarB类抗抑制蛋白 DarB-like antirestriction protein
73	46 436～50 134	1 232	-	GTG	DarB类抗抑制蛋白 DarB-like antirestriction protein
74	50 192～51 736	514	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
75	51 733～52 458	241	-	ATG	PAPS还原酶类蛋白 PAPS reductase-like protein
76	52 470～53 303	277	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
77	53 293～53 850	185	-	ATG	酰基辅酶A N-酰基转移酶 Acyl-CoA N-acyltransferase
79	54 246～55 979	577	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
80	55 991～56 977	328	-	ATG	尾纤蛋白 Tail fiber protein
81	57 063～58 382	439	-	ATG	推定尾纤蛋白 Putative tail fiber protein
82	58 382～58 663	93	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
84	60 366～61 343	325	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
85	61 350～61 772	140	-	ATG	病毒粒子相关噬菌体蛋白 Virion-associated phage protein
88	62 505～63 167	220	-	ATG	病毒粒子相关蛋白 Virion associated protein
90	63 713～64 150	145	-	GTG	DarB类抗抑制蛋白 DarB-like antirestriction protein
91	64 147～64 512	121	-	GTG	钼蝶呤结合蛋白 Molybdopterin-binding protein
92	64 534～64 657	40	-	TAT	细胞色素B Cytochrome B

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