The Passivation Effect of Yiduling on TMV in Tobacco Seedling Stage

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

Abstract

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

CLC Number:

S435.72

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.

Figures/Tables 9

References 24

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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