**3. Results and discussion**

#### **3.1. Effect of SAR induction on TMV lesion size and its distribution**

Data from Figs. 2, 3 and 4 are based on one representative experiment (Table 1) and serve as an example of three separate experiments. Mock inoculation with abrasive had no effect on lesion diameter and its distribution compared to control plants (Figs. 2, 3A, B and 4A, B). Induction of SAR was clearly manifested in differences of lesion development (Fig. 1) and in lesion size (Fig. 2) 4 days after challenge inoculation in all experiments. Although in control plants no significant differences were found between 5th and 6th leaf levels (Fig. 2), induction of SAR caused significant differences in degree of diminishing TMV lesion size between the 5th and 6th leaf levels. The effect of SAR induction often was more pronounced on the 5th leaf level than the 6th one (Fig. 2). Therefore, data for 5th and 6th leaves are presented separately in Figs. 2, 3 and 4. Generally, mean lesion diameter of leaves with SAR (0.528 and 0.659 mm for 5th and 6th leaf, respectively) was about half of the leaves from control plants (1.099 – 1.110 mm) (Fig. 2, Table 1).


1For abbreviations and explanations of treatments see Fig. 2.

2 Number of lesions.

**Table 1.** Test for normal distribution and lesion size data of TMV inoculated *Nicotiana tabacum* cv. Xanthi nc plants.

**3. Results and discussion**

mm) (Fig. 2, Table 1).

1For abbreviations and explanations of treatments see Fig. 2.

2

Number of lesions.

**3.1. Effect of SAR induction on TMV lesion size and its distribution**

366 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

Data from Figs. 2, 3 and 4 are based on one representative experiment (Table 1) and serve as an example of three separate experiments. Mock inoculation with abrasive had no effect on lesion diameter and its distribution compared to control plants (Figs. 2, 3A, B and 4A, B). Induction of SAR was clearly manifested in differences of lesion development (Fig. 1) and in lesion size (Fig. 2) 4 days after challenge inoculation in all experiments. Although in control plants no significant differences were found between 5th and 6th leaf levels (Fig. 2), induction of SAR caused significant differences in degree of diminishing TMV lesion size between the 5th and 6th leaf levels. The effect of SAR induction often was more pronounced on the 5th leaf level than the 6th one (Fig. 2). Therefore, data for 5th and 6th leaves are presented separately in Figs. 2, 3 and 4. Generally, mean lesion diameter of leaves with SAR (0.528 and 0.659 mm for 5th and 6th leaf, respectively) was about half of the leaves from control plants (1.099 – 1.110

**Treatments Leaf No. n1 Lesion diameter (mm) Normality test**

Control 5 343 0.25 2.01 1.099 0.9906 0.027 LR2 5 656 0.20 2.30 0.793 0.9396 < 0.001 LR2 + TMV 5 471 0.28 2.35 1.019 0.9747 < 0.001 LR4 5 428 0.29 2.27 1.024 0.9897 0.004 LR4 + TMV 5 187 0.22 1.32 0.525 0.8847 < 0.001 Mock 5 311 0.32 2.08 1.169 0.9929 0.150 SAR 5 226 0.23 1.31 0.528 0.8570 < 0.001 Control 6 132 0.43 1.80 1.110 0.9781 0.031 LR2 6 250 0.38 1.66 1.000 0.9835 0.005 LR2 + TMV 6 408 0.41 2.07 1.087 0.9851 < 0.001 LR4 6 145 0.46 1.96 1.182 0.9891 0.320 LR4 + TMV 6 145 0.29 1.35 0.666 0.9159 < 0.001 Mock 6 154 0.32 1.89 1.170 0.9889 0.266 SAR 6 103 0.25 1.55 0.659 0.9195 < 0.001

**Table 1.** Test for normal distribution and lesion size data of TMV inoculated *Nicotiana tabacum* cv. Xanthi nc plants.

Min Max Mean Shapiro-

Wilk's *<sup>w</sup> <sup>p</sup>*

**Figure 2.** Mean lesion size (and standard deviation) after challenge inoculation with TMV on *Nicotiana tabacum* cv. Xanthi nc plants at leaf level 5 and 6. The four bottom-most leaves of plants were first inoculated with TMV and chal‐ lenged on the 7th day. The four inoculated leaves were removed from the plants 2 and 4 days after inoculation (LR2+TMV and LR4+TMV, respectively). LR2 and LR4: the plants were not inoculated with TMV, but their leaves were removed at similar time intervals. Control: untreated plants; mock inoculated plants were treated with abrasive only; SAR: inoculated with TMV and challenged 7 days later without further treatments [1,12].

The distribution of TMV lesion size in most cases did not follow a normal distribution neither in control nor in leaves with SAR and other treatments as indicated by the results of Shapiro-Wilk *w* test (Table 1). Therefore, we used a statistical method suitable for comparison of nonnormally distributed data. Comparison of multiple sample means under heteroscedasticity also showed highly significant differences (P < 0.001) between control leaves and leaves with SAR both in the 5th and 6th leaf levels (Fig. 4A, B).

Supporting the above data, the distribution of lesion sizes in control leaves and in leaves with SAR was massively different both in 5th and 6th leaves (Fig. 3A, B). The lesion sizes in control plants showed a plateau-like distribution and covered a wide range from 0.25 to 2.01 mm and 0.43 to 1.80 mm on leaves 5 and 6, respectively (Table 1). The distribution of lesions in resistant leaves with SAR was completely different showing a peak at 0.3—0.8 mm range (about 70-80% of total number of lesions) and above 1.5 mm size, practically no lesions were detected (Fig. 3A, B, Table 1).

TMV causes local hypersensitive necrotic lesions in tobacco plants carrying *N* gene from *Nicotiana glutinosa* L. [20], accompanied by programmed cell death and development of symptoms within 2 days after inoculation. This resistant response is further strengthened during SAR induction as indicated by limited lesion size and a different type of lesion size distribution as compared to control plants. Similar to our results, a non-normal distribution of necrotic spots was reported in a resistant plant—fungus interaction [13]. This shift in lesion size distribution is probably due to biochemical responses that are manifested in more effective restriction of lesion development, multiplication/growth and/or movement of the pathogen in resistant genotypes. It has been reported recently that an antiviral agent against TMV can induce SAR in tobacco [9].

**Figure 3.** Kernel density estimation of TMV lesion size distribution on *Nicotiana tabacum* cv. Xanthi nc leaves at leaf level 5 (upper panel, A) and 6 (lower panel, B) after induction of systemic acquired resistance (SAR). For abbreviations and explanations of treatments see Fig. 2.

#### **3.2. Effect of sequential removal of inducing leaves on SAR development**

In order to detect the timing of signal transduction process from infected leaves to distant leaves, four bottom-most infected leaves were removed from tobacco plants at different time intervals, 2 (leaf removal, LR2) or 4 (LR4) days after TMV infection (Figs. 2, 3 and 4). The leaf removal without TMV infection after 4 days did not result in a significant shift in lesion development (LR4, Figs. 1 and 2, Table 1). Moreover, distribution of lesion sizes showed a plateau-like picture, comparable to control leaves (Fig. 3). Simultaneous comparison of treatments also showed that LR4 treated plants did not significantly differ from control plants at least on the 6th leaf level (p = 0.3500 with confidence interval: [-0.0353; 0.1805], Fig. 4B). On leaf level 5 the effect was almost the same as on level 6 indicating a limited effect (p = 0.0346 with confidence interval: [-0.146; -0.003], Fig.4A). On the contrary, removal of TMV-infected leaves after 4 days (LR4+TMV) mimicked the development of SAR in all characteristics in all three experiments. Lesion development was considerably, about 50% inhibited (Figs. 1, 2 and Table 1). The statistical analysis of data clearly showed highly significant differences between LR4+TMV and control plants (p < 0.001 for both leaf levels) but no significant differences between LR4+TMV and SAR treatments at both leaf levels (Fig. 4A, B). Not surprisingly, the distribution of lesion development of LR4+TMV plants was nearly the same as in leaves with SAR showing a characteristic peak at about 0.5 mm of lesion diameter. (Fig. 3A, B). These results clearly indicate that a 4-day period after the inducing infection of lower 4 leaves is enough for complete signal transduction of SAR in distant leaves. Consequently, the move‐ ment of signal molecule(s) should be detectable before this time point. These results also indicate that lesion size distribution as a resistance marker is a suitable tool for prediction of signalling events. Similar experiments with removal of leaves after a 2-day interval (LR2 and LR2+TMV) showed less clear evidences. Leaf removal without TMV infection (LR2) consid‐ erably influenced lesion development in all experiments (Fig. 2, Table 1) and somewhat shifted distribution of lesion size at both leaf levels (Fig. 3A, B). LR2 plants showed significant differences as compared either to control or SAR treated plants (Fig. 4A, B). This fact could be related to a different mechanism as compared to SAR induction, for example differences in hormone balance of distant leaves during longer incubation period after leaf removal. In LR2+TMV plants, the development of SAR was not detected on the basis of lesion size and its distribution characteristics (Figs. 2, 3A, B). Family-wise comparison of data rather suggests that LR2+TMV plants did not significantly differ from control ones but differed from the SAR treatment (p = 0.0316, confidence interval: [-0.1543; -0.0044]), Fig. 4A, B).

Altogether, these results indicate that the signal transduction starts probably only after visual appearance of local TMV symptoms (40—48 h post inoculation) and it is completed within the next 2 days. The identification of the exact timing of signal transduction from induced leaves is necessary for the further characterization of signal molecule(s) in phloem sap-enriched petiolar exudates.

In conclusion, we developed an easily applicable semi-automated method for the detection of the size of necrotic lesions and its distribution in tobacco leaves after TMV inoculation using appropriate statistical analysis. Decreased lesion size diameter and its characteristic nonnormal, right-skewed distribution seem to be an accurate and important feature of the resistant response in distant leaves with SAR. Application of this method during SAR induction indicated that signal transduction is completed in distant leaves by the 4th day after inducing TMV inoculation. Further experiments are in progress to characterize the chemical nature of this signal.

**3.2. Effect of sequential removal of inducing leaves on SAR development**

and explanations of treatments see Fig. 2.

368 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

In order to detect the timing of signal transduction process from infected leaves to distant leaves, four bottom-most infected leaves were removed from tobacco plants at different time intervals, 2 (leaf removal, LR2) or 4 (LR4) days after TMV infection (Figs. 2, 3 and 4). The leaf removal without TMV infection after 4 days did not result in a significant shift in lesion

**Figure 3.** Kernel density estimation of TMV lesion size distribution on *Nicotiana tabacum* cv. Xanthi nc leaves at leaf level 5 (upper panel, A) and 6 (lower panel, B) after induction of systemic acquired resistance (SAR). For abbreviations

**Figure 4.** Multiple comparisons of group means of selected treatments on leaf level 5 (A) and 6 (B). Dots represent the difference of the estimated means between treatments. Brackets flank the 95% confidence intervals. The difference is considered significant if the confidence interval does not contain the 0, represented by a vertical dashed line. Abbrevia‐ tions and explanations are the same as on Fig. 2.
