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In TMV preparations mixed with chitosan, we found that the virions were not infrequently associated with the polysaccharide particles (Figure 1).
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Both the infectivity and the viral coat protein content in leaves of N. tabacum L. cv. Samsun inoculated with TMV (2 μg/mL) mixed with chitosan (1 mg/mL) were significantly lower in the early period of infection than in leaves inoculated with TMV only. Thus, 3 d after inoculation, the infectivity (Table 1) and the viral coat protein content (Table 2) in the test leaf tissues were reduced by 63% and 66%, respectively, as compared to the control. Chitosan applied 24 h prior to inoculation with TMV also inhibited the infectivity (Table 1) and the viral coat protein content (Table 2) in the leaves, but to a lesser extent than when TMV and chitosan were inoculated together. As the infection developed, the inhibitory effects of the preparation decreased.
Days after infection Relative infectivity (%), TMV + chitosan Inhibitory effect (%) Relative infectivity (%), TMV after treatment with chitosan for 24 h Inhibitory ffect (%) 3 37 ± 4.1*** 63 59 ± 6.2*** 41 5 59 ± 6.0*** 41 74 ± 7.6** 26 7 74 ± 7.8** 26 82 ± 8.8* 18 TMV concentration, 2 μg /mL; chitosan concentration, 1 mg /mL. The relative infectivity is the number of local lesions per leaf half of N. tabacum L. cv. Xanthi-nc, expressed as a percentage of the untreated control. Each value is the average percentage obtained on 50 leaf halves of N. tabacum L. cv. Xanthi-nc (± standard error). The differences are statistically significant relative to controls at P* < 0.05, P** < 0.01 and P*** < 0.001. Table 1. The effect of chitosan on infectivity in N. tabacum L. cv. Samsun leaves infected with TMV
Days after infection Content of viral coat protein (ng/mm2) Inhibitory effect
(%)Content of viral coat protein (ng/mm2) Inhibitory effect
(%)TMV + chitosan Control TMV after treatment with chitosan for 24 h Control 3 1.5 ± 0.17*** 4.4 ± 0.5 66 2.1 ± 0.23*** 4.1 ± 0.42 49 5 6.1 ± 0.7** 11.5 ± 1.3 47 9.3 ± 0.94* 13.5 ± 1.45 31 7 18 ± 1.8* 25 ± 2.6 28 18.5 ± 1.9 22.3 ± 2.3 17 The viral coat protein content in test and control leaves was measured by ELISA and calculated for leaf disk areas of 1 mm2. Each value is the average of 10 replicates (± standard error). The differences are statistically significant relative to controls at P* < 0.05, P** < 0.01 and P*** < 0.001. Table 2. The effect of chitosan on viral coat protein content in N. tabacum L. cv. Samsun leaves infected with TMV
It should be noted that leaf halves of N. tabacum L. cv. Samsun inoculated with a mixture of TMV and chitosan and incubated in a humidity chamber did not show any toxicity symptoms and they appeared similar to leaf halves inoculated with TMV alone (data not shown).
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Inoculation of N. tabacum L. cv. Samsun leaves with a mixture of TMV and chitosan resulted in a significant increase in the activities of hydrolytic enzymes (proteases, RNases) in comparison to leaves inoculated with TMV only (Table 3).
Days after infection Hydrolases The activities of hydrolase (OD) TMV + chitosan TMV 3 Proteases 0.53 ± 0.05 0.38 ± 0.04 RNases 0.47 ± 0.05 0.32 ± 0.04 5 Proteases 0.50 ± 0.05 0.35 ± 0.04 RNases 0.38 ± 0.04 0.26 ± 0.03 7 Proteases 0.35 ± 0.04 0.23 ± 0.03 RNases 0.31 ± 0.03 0.21 ± 0.02 The activities of hydrolases were determined in 50 disks (4 mm in diameter) cut from leaf halves and expressed in terms of optical density. Each value is the average of 5 replicates (± standard error). The differences are statistically significant relative to controls at P < 0.05. Table 3. The activity of hydrolases in leaves N. tabacum L. cv. Samsun inoculated with TMV mixed with chitosan and inoculated with TMV alone
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In electron microscope assays of PTA-stained preparations from the infected leaves, we found that such preparations contained, along with normal TMV particles (18 nm in diameter, 300 nm long), abnormal virions, the diameter and length of which were substantially larger or smaller than the normal values (Figure 2). In PTAstained preparations from infected leaves previously fixed with glutaraldehyde, the ratio between normal and abnormal virions did not differ significantly from the ratio in preparations from leaves that had not been fixed with glutaraldehyde.
Figure 2. PTA-stained TMV particles from N. tabacum L. cv. Samsun leaf 3 d after inoculation with a mixture of TMV and chitosan. The medium-thickness arrows indicate virions of normal diameter (18 nm); thick arrows indicate swollen virions; thin arrows indicate thin virions. Asterisks show short TMV particles. Scale Bar = 100 nm.
An analysis of the distribution of TMV particle diameters (Table 4) showed that suspensions from the leaves inoculated with a mixture of TMV and chitosan contained a considerably greater number of abnormal virions than those from leaves inoculated with TMV only. Thus, in suspensions from leaves inoculated with a mixture of TMV and chitosan, 26% of viral particles were of normal diameter, 23% were swollen, and 51% were thin. In suspensions from infected untreated leaves, it was found that 63% of virions were of normal diameter, 13% were swollen, and 24% were thin. Calculation of the incidence of short virions showed that in suspensions from leaves infected with a mixture of TMV and chitosan, 40% of the virions were short, whereas in suspensions from control leaves, the incidence was 27%. Amongst the short viral particles, we sometimes observed particles of normal diameter, but much more frequently short particles with diameters greater or smaller than normal were seen.
Diameter of TMV particles, nm Number of TMV particles TMV + chitosan TMV 12 68 ± 7.0** 11 ± 2.5 14 104 ± 12** 22 ± 4.0 16 31 ± 4.0* 65 ± 7.0 18 105 ± 12** 251 ± 25 20 39 ± 5.0** 10 ± 1.5 22 24 ± 3.0 18 ± 2.0 24 29 ± 3.5 23 ± 3.0 The diameters of TMV virions were measured for 20 randomly selected viewing fields at a total magnification of x150000. Experiments were repeated three times. In each experiment, the diameters of 400 viral particles from the test sample and 400 particles from the control sample were measured. Each value is the average of three experiments (± standard error). The differences are statistically significant relative to controls at P* < 0.05 and P** < 0.01. Table 4. Diameter distribution of PTA-stained TMV particles in sap from N. tabacum L. cv. Samsun leaves 3 days after inoculation with TMV mixed with chitosan and with TMV alone
Immuno-electron microscopic studies showed that the virions of normal diameter, as well as the swollen viral particles, were completely decorated with TMV-specific antiserum while the thin virions lost the ability to bind to specific antibodies (Figure 3). In the virions of variable diameter, the antiserim decoration was observed only on the virion parts that were of comparatively large diameter (Figure 3). The antiserum to PVX did not cover the TMV particles.
Figure 3. TMV particles from N. tabacum L. cv. Samsun leaf 3 d after inoculation with a mixture of TMV and chitosan. A sap dip from the leaves was exposed to a TMV-specific antiserum and stained with PTA. The medium-thickness, thick, and thin arrows indicate normal, swollen, and thin virions, respectively. The arrowheads show antiserum. The virions of normal diameter, as well as the swollen virions, are completely covered with the antiserum. In the virions of variable diameter (at the bottom of the figure), the antiserum decoration is seen only on the virion parts that are of comparatively large diameter. Scale Bar = 100 nm.