Author: Margaret Kenny

Chemical Inactivation of Tobacco mosaic virus in Tobacco Seed
R.E.L. Loveday1, C.A. Wilkinson1, S.A. Tolin2, and P.J. Shepherd1
(1) Southern Piedmont Agricultural Research and Extension Center, Virginia Tech, Blackstone, VA
(2) Plant Pathology, Physiology, and Weed Science Department, Virginia Tech, Blacksburg, VA

1% HCl

Introduction
Tobacco mosaic virus (TMV) reduces tobacco (Nicotiana tabacum L.) yield and quality worldwide in all tobacco types.
TMV is located in the nucellar layer of the tobacco seed and is seed transmitted.
Chemical inactivation of TMV in the seed could reduce or eliminate an early season source of primary inoculum.

Objective
To determine the effectiveness of chemical treatments on infectious TMV in tobacco seed.

Materials and Methods cont.
Plants were rated 7 to 14 d after inoculation for the presence or absence of lesions.
An arcsine transformation was used for all data.
The healthy control was omitted from the analysis of percent TMV positive seedlings or seed coats.
Treatment means were separated using Duncan’s multiple range test.
All data presented are untransformed but Duncan’s multiple range test is from transformed analysis.

a

b

1% HCl

10% Na3PO4

10% Bleach

0.1% KMnO4

120g/L nonfat dry milk

2% CH2O

DI H2O infected control

DI H2O healthy control

Figure 5. Percent germination of chemically treated K 326 flue-cured tobacco seed grown in a growth chamber. Means with the same letter are not significantly different at the 0.05 probability level using Duncan’s mean separation.

0.2% HCl/10 min.

10% Na3PO4

10% Bleach

0.1% KMnO4

120g/L nonfat dry milk

2% CH2O

DI H2O infected control

Figure 8. Influence of chemical treatments on seed transmission of tobacco mosaic
virus in K 326 flue-cured tobacco grown in a growth chamber. The control treatment was excluded from the analysis.

DI H2O healthy control

0.2% HCl/10 min.

1% HCl

10% Na3PO4

10% Bleach

0.1% KMnO4

120g/L nonfat dry milk

2% CH2O

DI H2O infected control

DI H2O healthy control

Figure 11. Influence of chemical treatments on seed transmission of tobacco mosaic virus in K 326 flue-cured tobacco grown in a growth chamber. The control treatment was excluded from the analysis.

Summary
Hydrochloric acid and trisodium phosphate were the most effective in reducing seed transmission of TMV.
No chemical treatment eliminated the TMV antigen in the seed coat of seed collected from a TMV infected plant based on ELISA.
However, the virus was not biologically active in many infectivity assays.
Further research is needed to optimize the chemical, chemical rate, and treatment time.

0.5% HCl/10 min.

0.5% HCl/10 min.

0.2% HCl/10 min.

1% HCl/10 min.

1% HCl/10 min.

0.5% HCl/10 min.

Figure 2. Agitation of seed in chemical treatments (a) and air drying seed (b).

1% HCl/30 min.

10% Na3PO4/30 min.

1% HCl/30 min.

10% Na3PO4/30 min.

1% HCl/10 min.

15% Na3PO4/10 min.

1% HCl/30 min.

20% Na3PO4/10 min.

15% Na3PO4/10 min.

20% Na3PO4/10 min.

10% Na3PO4/30 min.

a

b

10% Bleach/30 min.

20% Bleach/10 min.

10% Bleach/30 min.

15% Na3PO4/10 min.

20% Na3PO4/10 min.

Figure 1. Tobacco mosaic virus symptoms on field grown K 326 flue-cured tobacco.

Materials and Methods
Seed were collected from field grown flue-cured tobacco cultivar K 326 with visible mosaic symptoms (Fig. 1).
Seed for the control treatment were from K 326 not infected with TMV.
Growth chamber tests were conducted at 22°C and a 12:12 h day:night cycle.
Tests were conducted in a randomized complete block design with three replications.
Each test was conducted twice.
Seed were agitated in chemical solutions at room temperature, rinsed, and air dried (Fig. 2).
Fifty treated seed were placed on water saturated blue blotter paper in rectangular clear plastic containers with lids.
Seedlings were separated from seed coats at 14 d (Fig. 3).
Enzyme linked immunosorbent assays (ELISA) were conducted using Agdia PathoScreen TMV-c kits (Fig. 4).
A sample was positive for TMV if it was yellow and the absorbance was twice that of the negative control.
Infectivity assays were conducted on Xanthi to determine if the virus was biologically active.

Figure 3. Germinated seedlings two weeks after seeding (a) and separating seedlings from seed coats (b).

Figure 4. Enzyme linked immunosorbent assay (ELISA) plate (yellow wells are positive for TMV).

Figure 6. Percent germination of chemically treated K 326 flue-cured tobacco seed grown in a growth chamber.

30% Bleach/10 min.

DI H2O healthy control/30 min.

a

ab

b

Figure 7. Percent germination of chemically treated K 326 flue-cured tobacco seed grown in a growth chamber.

20% Bleach/10 min.

Figure 9. Influence of chemical treatments on seed transmission of tobacco mosaic virus in K 326 flue-cured tobacco grown in a growth chamber. The control treatment was excluded from the analysis.

30% Bleach/10 min.

DI H2O healthy control/30 min.

Figure 10. Influence of chemical treatments on seed transmission of tobacco mosaic virus in K 326 flue-cured tobacco grown in a growth chamber. The control treatment was excluded from the analysis.

10% Bleach/30 min.

20% Bleach/10 min.

30% Bleach/10 min.

Figure 12. Influence of chemical treatments on seed transmission of tobacco mosaic virus in K 326 flue-cured tobacco grown in a growth chamber. The control treatment was excluded from the analysis.

DI H2O healthy control/30 min.

Results
A total of 4,709 seedlings in 332 samples and 9,495 seed coats in 210 samples were assayed from four tests.
Formaldehyde (CH2O) significantly reduced percent germination (Fig. 5).
The 1% hydrochloric acid (HCl) treatment significantly decreased percent germination (Fig. 6).
Percent germination was not significantly decreased with 0.2% or 0.5% HCl or 15% or 20% trisodium phosphate (Na3PO4) (Fig. 6).
Percent germination decreased with increasing treatment time (Fig. 7).
The 1% HCl treatment eliminated seed transmission of TMV in one test (Fig. 8) but not in the second test (Fig. 9).
Seed transmission of TMV was significantly reduced by treating with 10% Na3PO4 in one test (Fig. 8) and 20% Na3PO4 in a second test (Fig. 9).
Increasing treatment time did not significantly reduce seed transmission of TMV (Fig. 10).
All seed coat samples were positive for the TMV antigen (Fig. 11 and 12).

Figure 13. Tobacco mosaic virus symptoms on greenhouse tobacco transplants.

Acknowledgements
Financial support was provided by Philip Morris USA and Philip Morris International.

Carol A. Wilkinson wilki@vt.edu
P.J. Shepherd pjshep@vt.edu