抑毒灵对烟草育苗期TMV的钝化作用

doi:10.11924/j.issn.1000-6850.casb2021-0478

摘要/Abstract

摘要：

选用对烟草花叶病毒(tobacco mosaic virus,TMV)具有显著钝化作用的脱脂牛奶和磷酸三钠为研究对象,通过枯斑法测定脱脂牛奶和磷酸三钠两者复配制剂（称为抑毒灵）对烟草漂浮育苗过程中被TMV污染的基质及剪叶器的钝化作用,并利用透射电镜观察抑毒灵对病毒粒体的影响,同时采用实时qPCR对TMV含量以及寄主防御蛋白相对表达量进行定量检测。抑毒灵稀释液对TMV的钝化作用表现出时间和浓度依赖性,且不同处理间抑制率差异显著。抑毒灵与TMV接种液混合3 min后可完全抑制TMV对叶片的侵染。采用500倍抑毒灵消毒1 min以上时间抑制效果最佳。剪叶器具经稀释500倍的抑毒灵浸泡不少于5 min时对TMV的钝化效果可达100%。不同浓度的抑毒灵稀释液可显著钝化基质中的TMV,抑制TMV对烟苗的侵染和发病率,基质中TMV的含量下降74.09%~90.26%,相对防效达54.83%~85.45%。此外,所有抑毒灵处理中烟苗叶片表现正常,无药害发生。钝化病毒机制研究发现,经抑毒灵稀释液共孵育后的病毒粒体断裂成微小片段,外壳蛋白受损严重,螺旋结构和柱状基本消损完。与此同时,抑毒灵可提高防御酶PPO和PAL的酶活,还可显著诱导防御基因NPR1、PAL、PR1b和PR1a的表达。综上,抑毒灵可通过直接作用于病毒粒体和诱导寄主抗性起到抗病毒效果。

关键词: 烟草花叶病毒, 抑毒灵, 育苗期, 钝化作用, 防御蛋白, 寄主抗性

Abstract:

Skimmed milk and trisodium phosphate, which have a significant passivation effect on tobacco mosaic virus (TMV), were selected as the research objects. The passivation effect of the combination of skimmed milk and trisodium phosphate (called Yiduling) on TMV-contaminated substrate and leaf-cutting device during floating seedling cultivation was determined by the dead spot method. The effects of Yiduling on viral granules were observed by transmission electron microscopy. At the same time, real-time qPCR was used to quantitatively detect the content of TMV and the relative expression of host defense protein. The results showed that the passivation effect of Yiduling diluent on TMV was time and concentration dependent, and the inhibition rate was significantly different between different treatments. Yiduling mixed with TMV inoculum for 3 minutes completely inhibited the infection of TMV on leaves. The best inhibitory effect was obtained when 500 times Yiduling was used for disinfection for more than 1 min. The passivation effect on TMV was up to 100% when the leaf-cutting device was soaked with Yiduling diluted 500 times for no less than 5 minutes. Different concentrations of Yiduling diluent significantly inactivated the TMV in the substrate and inhibited the infection and morbidity of tobacco seedlings by TMV, and the content of TMV in the substrate decreased by 74.09%-90.26% and the relative control effect reached 54.83%-85.45%. In addition, the tobacco seedling leaves in all treatments with Yiduling were normal and no phytotoxicity occurred. The study of the mechanism of inactivating virus found that the virions after co-incubation with Yiduling diluent were broken into tiny fragments, the coat protein was severely damaged, and the helical structure and columnar shape were basically lost. At the same time, Yiduling increased the enzyme activity of defense enzymes PPO and PAL, and significantly induced the expression of defense genes NPR1, PAL, PR1b and PR1a. In summary, Yiduling can have antiviral effects by directly acting on virion and inducing host resistance.

Key words: tobacco mosaic virus (TMV), Yiduling, seedling stage, passivation effect, defense protein, host resistance

中图分类号:

S435.72

冯永新, 谭宏祥, 关辉, 靳彦峰, 徐伟, 王杰. 抑毒灵对烟草育苗期TMV的钝化作用[J]. 中国农学通报, 2022, 38(10): 112-120.

FENG Yongxin, TAN Hongxiang, GUAN Hui, JIN Yanfeng, XU Wei, WANG Jie. The Passivation Effect of Yiduling on TMV in Tobacco Seedling Stage[J]. Chinese Agricultural Science Bulletin, 2022, 38(10): 112-120.

图/表 9

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基因	引物序列	NCBI登录号
β-actin	F：5′-TCTTGACTACGAGCAGGAGC-3′	GQ339768.1
β-actin	R：5′-ATCAGCAATACCAGGGAACA-3′	GQ339768.1
TMVCP	F：5′-CCATCACAGTTCGTGTTCTTG-3′	EF183504.1
TMVCP	R：5′-TAGCGTCTAACGTTTCGGCAG-3′	EF183504.1
PR1-a	F：5′-TAACTCGGTTCGTGTTGGAT-3′	X12485.1
PR1-a	R：5′-TTAGTATGGACTTTCGCCTCT-3′	X12485.1
PR1-b	F：5′-TTTGGATGCCCATAACACA-3′	X12486.1
PR1-b	R：5′-AATCGCCACTTCCCTGA-3′	X12486.1
NPR1	F：5′-TGGAGCAAGCAGAAAGAAG-3′	KY402167.1
NPR1	R：5′-GCCAGTGGGAACTCAGAAG-3′	KY402167.1
PAL	F：5′-GTGAAGGCGGCTCAGAAG-3′	AB008199.1
PAL	R：5′-GTTGCCACCGTGTAATGC-3′	AB008199.1

基因	引物序列	NCBI登录号
β-actin	F：5′-TCTTGACTACGAGCAGGAGC-3′	GQ339768.1
β-actin	R：5′-ATCAGCAATACCAGGGAACA-3′	GQ339768.1
TMVCP	F：5′-CCATCACAGTTCGTGTTCTTG-3′	EF183504.1
TMVCP	R：5′-TAGCGTCTAACGTTTCGGCAG-3′	EF183504.1
PR1-a	F：5′-TAACTCGGTTCGTGTTGGAT-3′	X12485.1
PR1-a	R：5′-TTAGTATGGACTTTCGCCTCT-3′	X12485.1
PR1-b	F：5′-TTTGGATGCCCATAACACA-3′	X12486.1
PR1-b	R：5′-AATCGCCACTTCCCTGA-3′	X12486.1
NPR1	F：5′-TGGAGCAAGCAGAAAGAAG-3′	KY402167.1
NPR1	R：5′-GCCAGTGGGAACTCAGAAG-3′	KY402167.1
PAL	F：5′-GTGAAGGCGGCTCAGAAG-3′	AB008199.1
PAL	R：5′-GTTGCCACCGTGTAATGC-3′	AB008199.1

处理	稀释倍数	混合1 min		混合3 min
处理	稀释倍数	枯斑数/个	抑制率/%	枯斑数/个	抑制率/%
抑毒灵	500倍	3.00	99.08±1.08a	0.00	100.00±0.00 a
	800倍	79.40	75.60±2.79b	42.60	86.71±3.25 b
	1000倍	98.90	69.62±3.15c	80.50	74.89±2.14 c
PBS+TMV		325.50		320.60
PBS		0		0

处理	稀释倍数	混合1 min		混合3 min
处理	稀释倍数	枯斑数/个	抑制率/%	枯斑数/个	抑制率/%
抑毒灵	500倍	3.00	99.08±1.08a	0.00	100.00±0.00 a
	800倍	79.40	75.60±2.79b	42.60	86.71±3.25 b
	1000倍	98.90	69.62±3.15c	80.50	74.89±2.14 c
PBS+TMV		325.50		320.60
PBS		0		0

处理	稀释倍数	发病率/%			相对防效/%
处理	稀释倍数	3 min	5 min	10 min	3 min	5 min	10 min
抑毒灵	500倍	6.35±0.25d	0.00±0.00d	0.00±0.00d	77.30±0.07a	100.00±0.00a	100.00±0.00a
	800倍	10.79±0.53c	5.59±0.07c	1.24±0.04c	65.43±0.03b	86.34±0.02b	91.32±0.10b
	1000倍	15.66±0.79b	11.56±0.09b	4.64±0.09b	51.42±0.12c	65.24±0.11c	88.05±0.12c
二氧化氯	800倍	10.05±0.75c	6.03±0.12c	1.57±0.10c	60.49±0.13b	79.77±0.12b	85.66±0.19c
PBS+TMV		35.62±1.23a	35.79±1.05a	35.65±0.98a
PBS		0	0	0