**2. Materials and methods**

*Nicotiana tabacum* L. cv. Xanthi nc plants were grown in greenhouse and inoculated with TMV U1 strain at 6 leaf stage (fully expanded leaves) as we have described earlier [12]. The four bottom-most leaves of plants were inoculated for induction of systemic acquired resistance without any further treatments (treatment: SAR) [1]. A subset of these plants was further treated: the inoculated leaves were removed from the plants 2 and 4 days after inoculation (treatments: leaf removal, LR2+TMV and LR4+TMV, respectively). As a control, leaves were removed similarly from uninoculated plants (treatments LR2 and LR4). Seven days after inoculation, the two fully expanded distant leaves (the 5th and 6th leaf levels) were used for challenge inoculation and data analysis. Abrasive (carborundum, 50 mg/100ml) was added to the inoculum. Mock inoculation was performed only with abrasive in 10 mM K-phosphate buffer (pH = 7.0).

including hormonal compounds (salicylic acid and methyl jasmonate), benzothiadiazole, Ningnanmycin (an antiviral agent against TMV) and insect eggs have been shown to induce SAR [5-9]. The signal transduction process and its timing are essential components of SAR induction that moves from induced to distant leaves. Recent studies indicated that the

InductionofSARisoftenvalidatedbytheincreasedexpressionofmarkergenes indistanttissues and/or by limited symptom expression/multiplication of the pathogen. However, the exact evaluation of symptom expression often has serious limitations. Visual assessment ofleaf spots may lead to false analysis [11]. The number of developing TMV lesions is influenced by many factors (especially by the inoculation method and physiological state of plants) and therefore not well suitable as a single factor for characterization of the development of SAR [1,12].

In the present contribution we analysed local TMV lesion size formation and its distribution after SAR induction by a computer-assisted method. The method can detect nearly all lesions on tobacco leaves including the smallest ones (ca. 0.2 mm) and consequently is suitable for the exact determination and comparison of lesion size distribution of differentially treated plants. It is particularly important if a certain component is not normally distributed and mean of data is masking the fine differences. Sherwood [13] rejected normal distribution of fungal lesions (orchardgrass—*Stagonospora arenaria* interaction) in resistant genotypes, but not in susceptible ones. However, to the best of our knowledge, there is no systematic research on lesion size

**a.** To develop a reliable and well-adaptable method for semi-automated measurement of lesion size and its distribution using an appropriate statistical analysis (see Materials and

**c.** To determine the timing of signal transduction process(es) by comparing lesion size distributions of distant leaves after removal of TMV-infected signal-inducing leaves at

*Nicotiana tabacum* L. cv. Xanthi nc plants were grown in greenhouse and inoculated with TMV U1 strain at 6 leaf stage (fully expanded leaves) as we have described earlier [12]. The four bottom-most leaves of plants were inoculated for induction of systemic acquired resistance without any further treatments (treatment: SAR) [1]. A subset of these plants was further treated: the inoculated leaves were removed from the plants 2 and 4 days after inoculation (treatments: leaf removal, LR2+TMV and LR4+TMV, respectively). As a control, leaves were removed similarly from uninoculated plants (treatments LR2 and LR4). Seven days after inoculation, the two fully expanded distant leaves (the 5th and 6th leaf levels) were used for challenge inoculation and data analysis. Abrasive (carborundum, 50 mg/100ml) was added to

**b.** To evaluate and compare TMV lesion size distribution in control versus SAR plants

movement of SAR signal(s) is connected to plasmodesmata [10].

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

distribution of viral local necrotic infections.

Therefore, our tasks were as follows:

different time intervals

**2. Materials and methods**

methods)

Four days after challenge inoculation two or three detached leaves (of the same leaf level) per treatment, were directly scanned to obtain high resolution (300 dpi) digital images with a scanner (HP Scanjet G2k710). Data were collected from these images after threefold magnifi‐ cation on the computer screen. All dark, brownish black, round-shaped TMV lesions typical of this infection on tobacco plants were selected visually for analysis. The ImageJ 1.48v image analysis software [14] was used for lesion selection and size calculations. However, due to the low contrast of small TMV spots, lesion selection was done manually using a drawing tablet that outperforms the precision of a standard computer mouse. Lesion size was expressed as the mean of the major and minor axes of the best fitting ellipse having equal area to the lesion. Considering all lesions per leaf resulted in more accurate estimation of lesion size compared to former approaches [7,12,13]. Figure 1 demonstrates this process on TMV-infected tobacco leaves.

All calculations (lesion size distribution, mean and variance of lesion size) were carried out with R [15]. Shapiro-Wilk *w* test for normal distribution and density estimation of data were calculated with functions 'shapiro.test' and 'density', respectively with their default settings.

For comparison of sample means a multiple comparison procedure was used with the R package multcomp [16]. The method allows simultaneous comparisons while the family-wise error rate, used as the standard measure for false positive results in multiple testing, remains well controlled [17]. The method uses the HC3 covariance estimation [18,19]. Furthermore, this method tolerates unequal variances, non-normal distribution of data and unbalanced group sizes, which often occur in biological datasets.

**Figure 1.** Leaf necrosis on *Nicotiana tabacum* cv. Xanthi nc leaves after challenge inoculation. (A): control, (B): SAR, (C): the plants were not inoculated with TMV, but their leaves were removed after 4 days (LR4), (D): the four inoculated leaves were removed from the plants 4 days after inoculation (LR4+TMV). Bar: 3 cm. Red circles on (A) mark the proc‐ essed lesions during image analysis.
